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Abstract:

The present invention provides proteins with antimicrobial activity, and
methods for treating subjects by administering the proteins. In
particular, the invention provides methods for treating and/or preventing
microbial diseases and infections. The present invention further provides
the target for these antimicrobial agents, as well as assays for
identifying regulators of the target.

Claims:

1. A method of neutralizing spores of a prokaryotic pathogenic organism,
said method comprising contacting said pathogenic organisms with a
composition comprising an interferon-inducible (ELR-) CXC chemokine.

3. The method of claim 1 wherein the spores are from an organism selected
from the group consisting of Bacillus anthracis, Bacillus cereus,
Clostridium difficile, Clostridium botulinum, Clostridium perfringens,
Clostridium tetani and Clostridium sordellii.

4. The method of claim 3 wherein the spores are Bacillus anthracis or
Clostridium difficile spores.

11. The method of claim 5 wherein said composition further comprises a
lipid vesicle and said peptide or peptidomimetic derivative is
encapsulated within the lipid vesicle, or linked to the surface of said
lipid vesicle.

12. The method of claim 11 wherein said composition further comprises a
supplemental anti-microbial agent.

13. The method of claim 12 wherein the supplemental anti-microbial agent
is an antibiotic.

19. The antimicrobial composition of claim 14 wherein said composition
further comprises a lipid vesicle and said peptide or peptidomimetic
derivative is encapsulated within the lipid vesicle, or linked to the
surface of said lipid vesicle.

20. The antimicrobial composition of claim 19 wherein said composition
further comprises a supplemental anti-microbial agent.

21. (canceled)

22. A pharmaceutical composition comprising the non-native peptide of
claim 14; and a pharmaceutically acceptable carrier.

23. (canceled)

24. The method of claim 6 wherein said spores of the prokaryotic
pathogenic organism have colonized a host organism and have entered into
a stationary growth phase.

[0002] The sequence listing with file name SEQList_ST25.txt, created on
Feb. 18, 2011 (32.9 KB) is expressly incorporated by reference in its
entirety.

BACKGROUND

[0003] Pathogenic microbes are increasingly becoming resistant to
established antibiotic drugs. Only three new structural classes of
antibiotics have been introduced into medical practice in the past 40
years and certain pathogenic bacteria have become resistant to all these
classes. Moreover, all antimicrobial drugs on the market have some
relative degree of host toxicity that is concentration dependent.

[0004] B. anthracis is a Gram-positive, spore-forming bacterium that is
the causative agent of anthrax. There are three clinical forms of anthrax
that reflect the route by which the bacterial spores are introduced in
the host: cutaneous, gastrointestinal, and inhalational. Inhalational
anthrax is a disease that has been described as a biphasic clinical
illness characterized by a 1- to 4-day initial phase of malaise, fatigue,
fever, myalgias, and nonproductive cough. The initial phase is then
followed by a rapidly fulminant phase of respiratory distress, cyanosis,
and diaphoresis. Death typically follows the onset of the fulminant phase
in 1 to 2 days. Inhalational anthrax typically causes severe necrotizing
pneumonia, mediastinal invasive disease with resultant massive
hemorrhagic mediastinitis and lymphadenitis, and dissemination to other
organs, including the central nervous system, gastrointestinal tract,
lymph nodes, and vascular system. Since the initial phase of illness can
be confused with a non-specific viral respiratory infection, a diagnosis
of anthrax is often not entertained. Mortality is high (>85%) if the
diagnosis is delayed. In fact, even in the 2001 human anthrax cases
mortality was still high (45%) in spite of early recognition of
inhalational anthrax infection in the index case, early awareness of the
nefarious distribution of anthrax spores through the United States postal
system, and alerts raised amongst health care providers, leading to
empirical pre-emptive antibiotic intervention.

[0005] Because B. anthracis exists in two distinct forms (spores and
vegetative cells, i.e., bacilli) and causes human pulmonary infection
with disseminated disease, studies on B. anthracis have a broader
applicability for understanding host-pathogen interactions and host
response to bacterial pulmonary pathogens. The spores of B. anthracis are
the infectious form of the organism and are responsible for initiating
all forms of clinical anthrax. Spores are extremely hardy and can
withstand extremes of heat, mechanical disruption, ultraviolet
irradiation, and lytic enzymes. Spores are comprised of multiple
protective layers that consist, from the inside to the outside, of a
nucleic acid core surrounded by an inner spore membrane, cortex, outer
spore membrane, spore coat, and exosporium.

[0006] The dominant model of inhalational anthrax involves the uptake of
spores by alveolar macrophages or other phagocytic cells with subsequent
transport by the phagocytic cells to the mediastinal lymph nodes. Spore
germination and outgrowth of vegetative bacilli occur primarily in the
host cell cytosol, and the organisms eventually escape from the host cell
and disseminate throughout the host. All known virulence factors of B.
anthracis are produced by the vegetative bacilli. The virulence factors
include two bipartite toxins (lethal toxin and edema toxin) and a
poly-gamma-D-glutamic acid capsule.

[0007] The B. anthracis Ames strain possesses two plasmids that encode the
genes for the synthesis of the toxins and capsule (plasmids pXO1 and
pXO2, respectively). Sterne strain possesses pXO1 but not pXO2; thus,
Sterne strain is a toxigenic, unencapsulated B. anthracis strain. The
majority of our data has been generated using Sterne strain, although
select experiments have also been performed with Ames strain to confirm
that Sterne strain is a suitable model organism for our studies.

[0008] Much work has been devoted in the anthrax field towards
understanding critical host factors in anthrax infection. In vitro
experiments and in vivo work in mice have revealed the genetic locus for
Nalp1b appears to be a determinant of susceptibility, and defects in
Nalp1b in mouse macrophages lead to decreased in vitro release of the
pro-inflammatory cytokine, IL-1β. Furthermore, there are distinct
differences in mouse strain susceptibility to anthrax. For example, the
CS-deficient A/J mice are highly susceptible to inhalational (or
subcutaneous) infection with B. anthracis Sterne strain. In contrast, the
majority of mouse strains tested, including C57BL/6 mice, are resistant
to inhalational anthrax infection with B. anthracis Sterne strain.
However, C57BL/6 mice are susceptible to B. anthracis administered by
other routes of inoculation (e.g., subcutaneous injection), which would
suggest that important host defense factors are present or generated in
the lungs that are otherwise bypassed when the organisms are introduced
by another route.

[0009] In terms of the human host, susceptibility factors have not been
identified although age and diminished host immune response are likely
candidates, based on the observation that mortality from inhalational
anthrax in the 1972 accidental release of spores in the city of
Sverdlovsk in the former Soviet Union, as well as the 2001 anthrax cases
that occurred from intentional delivery of spores through the U.S. postal
system, occurred solely in adults and primarily, adults over the age of
35.

[0010] In a follow-up study of persons exposed to B. anthracis spores in
the U.S. Capitol building in 2001, adults in the "definite exposure"
group (based on exposure zone and positive nasopharyngeal swab cultures
for B. anthracis), but who did not subsequently develop or succumb to
clinical anthrax, had markers indicative of a cell-mediated immune
response with elevated levels of TNF-α, IL-1β, IL-6, and
CXCL9. Although it remains unclear which host factors play a role in
susceptibility in humans, an early cell-mediated host immune response is
likely a critical factor in this pulmonary infection that has a rapidly
fatal course if untreated, especially given that there is no time for the
host to mount an antibody-mediated immune response. An unfortunate twist
is that lethal toxin and to a lesser extent, edema toxin are now
recognized to play an important role in altering the host immune
response--this is an evolving story of toxin-mediated host immune
suppression, and the effects appear to be quite complex, depending on
whether one is studying toxin effects in vitro or in vivo. It appears,
however, that B. anthracis may be particularly skillful at surviving in
the host, likely through toxin suppression of the immune response.
Additionally, bacterial toxins are not affected by antibiotics and as
such, once the toxins are being produced, the window of opportunity
rapidly closes for containing and fighting this infection with standard
treatment modalities (i.e., antibiotics).

[0011] Another critically important issue is that B. anthracis, like many
other organisms, can acquire or develop antibiotic resistance such that
antibiotic choices will become limited. Thus, critically important
factors that can facilitate successful recovery of the host from this
infection include a combination of appropriate therapeutic intervention
plus an effective host immune response.

[0012] Chemokines are chemotactic cytokines that are important regulators
of leukocyte-mediated inflammation and immunity in response to a variety
of diseases and infectious processes in the host. Chemokines are a
superfamily of homologous 8-10 kDa heparin-binding proteins, originally
identified for their role in mediating leukocyte recruitment.

[0013] The four major families of chemokine ligands are classified on the
basis of a conserved amino acid sequence at their amino terminus, and are
designated CXC, CC, C, and CX3C sub-families (where "X" is a nonconserved
amino acid residue; reviewed in references 76, 78).

[0014] The interferon-inducible (ELR-) CXC chemokines are one of the
largest families of chemokines, and each member of this group contains
four cysteine residues. Most chemokines are small proteins (8-10 kDa in
size), have a net positive charge at neutral pH, and share considerable
amino acid sequence homology. Structurally, the defining feature of the
CXC chemokine family is a motif of four conserved cysteine residues, the
first two of which are separated by a non-conserved amino acid, thus
constituting the Cys-X-Cys or `CXC` motif This family is further
subdivided on the basis of the presence or absence of another three amino
acid sequence, glutamic acid-leucine-arginine (the `ELR` motif),
immediately proximal to the CXC sequence (see references 75, 119). The
ELR- positive (ELR+) CXC chemokines, which include IL-8/CXCL8, are potent
neutrophil chemoattractants and promote angiogenesis. Among the ELR-
negative (ELR-) CXC chemokines, CXCL9, CXCL10 and CXCL11, are potently
induced by both type 1 and type 2 interferons (IFN-α/β and
IFN-γ, respectively). These Interferon-inducible (ELR-) CXC
chemokines are generated by a variety of cell types (including monocytes,
macrophages, lymphocytes, and epithelial cells), and are extremely potent
chemoattractants for recruiting mononuclear leukocytes, including
activated Th1 CD4 T cells, natural killer (NK) cells, NKT cells, and
dendritic cells to sites of inflammation and inhibiting angiogenesis.

[0015] The chemokine receptors are a family of related receptors that are
expressed on the surface of all leukocytes. The shared receptor for
CXCL9, CXCL10, and CXCL11 is CXCR3 (see references 69, 72, 92, 97, 111).
Through their interaction with CXCR3, the ligands CXCL9, CXCL10 and
CXCL11 are the major recruiters of specific leukocytes, including CD4 T
cells, NK cells, and myeloid dendritic cells. Importantly, this chemokine
ligand-receptor system is at the core of a positive feedback loop
escalating Th1 immunity, whereby cytokines such as interleukin (IL)-12
and IL-18 (released by myeloid accessory cells) activate local NK cells
to produce IFN-γ, which then induces generation of CXCL9, CXCL10,
and CXCL11, which then recruits CXCR3-expressing cells that act as a
further source of IFN-γ, which then induces further production of
CXCL9, CXCL10, and CXCL11. Consistent with the importance of these
interferon-inducible (ELR-) CXC chemokines in promoting Thl-mediated
immunity, CXCR3 and its ligands have been documented to play a critical
role in host defense against many micro-organisms, including viruses,
Mycobacterium tuberculosis, other bacteria, and protozoa.

[0016] Independent of their role in CXCR3-dependent leukocyte recruitment,
CXCL9, CXCL10, and CXCL11 have recently been found to display direct
antimicrobial properties that resemble those of defensins (see references
33, 40). These antimicrobial effects were first demonstrated in 2001
against Escherichia coli and Listeria monocytogenes. Subsequently, an
increasing number of chemokines have been shown to have antimicrobial
activity against various strains of bacteria and fungi, including E.
coli, S. aureus, Candida albicans, and Cryptococcus neoformans (see
references 112, 114).

[0017] There is a long felt need in the art for new compositions and
methods useful as antimicrobial agents, as well as targets for
antimicrobial agents. The present invention satisfies these needs.

SUMMARY OF THE INVENTION

[0018] The present disclosure provides methods for treating and/or
preventing microbial diseases. The invention also provides methods for
treating and/or preventing microbial infections. In accordance with one
embodiment compositions comprising interferon-inducible (ELR-) CXC
chemokines, including for example chemokines CXCL9, CXCL10 and CXCL11,
can be used to neutralize actively growing, as well as stationary phase,
pathogenic bacteria. Furthermore, the chemokine compositions of the
present invention have been discovered to be surprisingly effective in
neutralizing the spores of pathogenic bacteria, including spores of
Bacillus anthracis. The compositions disclosed herein can be used as a
therapeutic intervention and innovative approach for treating pulmonary
and gastrointestinal bacterial pathogens, especially at a time when it is
becoming increasingly clear that expanding antibiotic resistance in
bacterial pathogens is moving the medical field into a post-antibiotic
era.

[0019] In some embodiments, the methods of the invention comprise
administering to a subject a therapeutically effective amount of at least
one compound of the invention. In one aspect, the compound is a peptide,
or a fragment, homolog, or modification thereof. In one aspect, an
isolated nucleic acid comprising a nucleic acid sequence encoding a
peptide of the invention is administered.

[0020] The present invention encompasses the theory disclosed herein that,
inter alia, interferon-inducible (ELR-) CXC chemokines exhibit
antimicrobial activity. In one aspect, the microbes are bacteria. In
another aspect, the bacteria include Gram-positive and Gram-negative
bacteria.

[0021] It is also disclosed herein that FtsX is the putative bacterial
target for interferon-inducible (ELR-) CXC chemokines in B. anthracis.
The present invention therefore encompasses targeting FtsX either
directly or indirectly for use as an antimicrobial agent or target of an
antimicrobial agent. The present invention further provides compositions
and methods useful for identifying regulators of FtsX, and therefore,
identifying antimicrobial agents. In one aspect, the present invention
provides compositions, methods, and assays utilizing FtsX to identify
compounds that regulate FtsX function or levels or downstream activity.
In one aspect, the regulation is inhibition. In one aspect, compounds
identified in these assays exhibit anti-microbial activity as described
herein. The types of compounds useful in the invention include, but are
not limited to, proteins and peptides, as well as active fragments and
homologs thereof, drugs, and peptide mimetics. In one aspect, the active
fragments, homologs, and mimetics are fragments, homologs, and mimetics
or agonists of the chemokines described herein.

[0022] It is disclosed herein that FtsX is a target of
interferon-inducible (ELR-) chemokines and that these chemokines have
antimicrobial activity against bacteria expressing FtsX. Therefore, the
present invention encompasses the use of isolated FtsX as a vaccine or
therapeutic immunogenic agent useful for preventing or treating
infections or diseases involving FtsX-expressing microbes. In one aspect,
an isolated nucleic acid comprising a sequence encoding FtsX or a
fragment or homolog thereof can be administered to a subject in need
thereof In another aspect, an immunogenic amount of an isolated FtsX
protein, or a fragment of homolog thereof can be administered to a
subject in need thereof.

[0023] Further embodiments of the invention include therapeutic kits that
comprise, in suitable container means, a pharmaceutical formulation of at
least one antimicrobial peptide of the invention. Some embodiments
provide kits comprising a pharmaceutical formulation comprising at least
one peptide of the invention and a pharmaceutical formulation of at least
one antimicrobial agent or antibiotic. The antimicrobial peptide and
antimicrobial agent or antibiotic may be contained within a single
container means, or a plurality of distinct containers may be employed.

[0024] Various aspects and embodiments of the invention are described in
further detail below.

[0030] FIGS. 4A & 4B. Isolation and confirmation of CXCL10-resistant
isolates from B. anthracis transposon mutagenesis library screen. Using a
mariner transposon mutagenesis library of B. anthracis Sterne strain, a
pool of vegetative cells grown from the library (>50,000 CFU's,
representing ˜10× genome coverage) was incubated with 48
μg/ml CXCL10 or buffer only (untreated) for 1 hr at 37° C.
Vegetative cells were plated onto BHI plates+erythromycin (selection
marker for library). For untreated cells, a lawn of colonies was
obtained, but for a CXCL10-treated library, 13 colonies were obtained
from one screen (FIG. 4A), and a total of 18 colonies were obtained from
two separate screens. Each of the 18 isolates (TNX1-18) was tested for
resistance to CXCL10 using an Alamar Blue assay (FIG. 4B).
**p-value<0.01, ***p-value<0.001 compared to the B. anthracis
Sterne strain 7702 (wildtype strain, designated "7702") and compared to
CXCL10-treated library (designated "Library"); n.d.=not detectable.

[0031] FIGS. 5A & 5B. Schematic of prototypical ABC transporters that
function as importers or exporters. The prototype in Gram-negative
bacteria is highlighted in the schematic drawing of FIG. 5A and the
prototype in Gram-positive bacteria is highlighted in the schematic
drawing of FIG. 5B. The typical components of the ABC transporter consist
of a substrate binding protein (SBP), a membrane-spanning domain (MSD) as
a heterodimer, and an ATPase or nucleotide binding protein (NBP). Figure
taken from Braibant M., P. Gilot, and J. Content (2000) FEMS Microbiol.
Rev. 24:449-467.

[0032]FIG. 6. Predicted topology of the B. anthracis FtsX, generated
using program software available at the website for Center for Biological
Sequence Analysis of the Technical University of Denmark. Negatively- and
positively-charged amino acids are shaded, and the negatively-charged
amino acids are designated with an asterisk.

[0033]FIG. 7. B. anthracis ftsX mutant strain is resistant to CXCL10.
Susceptibility to human CXCL10 (48 ug/ml for 6 hr) was tested using an
Alamar Blue assay. Strains tested were: the transposon mutagenesis
library, TNX18 isolated from the screen, and the B. anthracis ftsX mutant
strain (with ftsX deleted; this strain is also designated in the text as
4ftsX strain). Both TNX18 and the ftsX mutant strain exhibited resistance
to CXCL10.

[0034]FIG. 8. The B. anthracis ftsX mutant is also resistant to CXCL9 and
CXCL11. Susceptibilities to human CXCL9 and CXCL11 (48 ug/ml for 6 hr
each) were tested using an Alamar Blue assay. Strains tested were: B.
anthracis Sterne strain 7702 parent strain, transposon mutagenesis
library, TNX18, and the ftsX mutant. TNX18 and the ftsX mutant were
resistant to CXCL9. All strains were resistant to CXCL11, which is
consistent with the less effective antimicrobial activity observed for
human CXCL11.

[0035]FIG. 9. Neutralization of CXCL9, CXCL9/CXCL10, or CXCL9/10/11 but
not CXCR3 renders C57BL/6 mice susceptible to B. anthracis infection.
C57BL/6 mice received injections of anti-CXCL9, CXCL10, and/or CXCL11
antibodies or anti-CXCR3 antibodies or control goat serum, as indicated
in the figure, one day prior to intranasal inoculation with B. anthracis
Sterne strain spores and then daily throughout the experiment. Mice were
monitored for survival over an 18-day period. *p-value<0.05;
**p-value<0.01 compared to spore-inoculated animals that received
control goat serum.

[0036] FIGS. 10A & 10B Susceptibility of B. anthracis Sterne strain 7702
spores to CXCL10. By treating B. anthracis cultures with an
interferon-inducible (ELR-) CXC chemokine in the presence and absence of
a heat treatment, one can determine the effectiveness of the
interferon-inducible (ELR-) CXC chemokine on spore viability. Thererfore,
CFU counts were determined for B. anthracis in the presence and absence
of heat treatment at 65° C. for 30 minutes. Cultures not exposed
to heat treatment when plated will indicate the number of vegetative and
viable spores that were present in the culture, whereas the heat treated
culture will only produce CFUs representative of the number of viable
spores that were in the culture. As shown in FIG. 10A, treatment with
human CXCL9, CXCL10 or CXCL11 (48 ug/ml each for 6 hr) reduced (CXCL11)
or eliminated (CXCL9, CXCL10) vegetative outgrowth and disrupted spore
germination (CXCL9, CXCL 10). Similar results were obtained for murine
interferon-inducible (ELR-) CXC chemokines (see FIG. 10B using an Alamar
Blue assay).

[0037] FIG. 11A-11C. Susceptibility of exponential versus stationary phase
B. anthracis Sterne strain 7702 to CXCL10. Overnight cultures were either
diluted back in fresh medium and grown to exponential phase prior to
addition of buffer control or CXCL10 at 8 μg/ml (ie, ˜EC50
value; for exponential phase organisms as shown in FIG. 12B below) or
used directly from overnight cultures by spinning down, reconstituting in
same volume fresh medium plus buffer control or CXCL10 at 8 μg/ml.
Aliquots were plated out for CFU determination after an incubation of 30
min or 1 hr. The data from exponential phase B. anthracis are shown in
FIG. 11A and data from stationary phase B. anthracis are shown in FIG.
11B. A concentration curve for CXCL10 against stationary phase organisms
is shown in (FIG. 11C) with an EC50 value determined to be 0.33
+/-0.05 μg/ml. Each experiment was performed 3 separate times in
triplicates. n.d., not detected.

[0062] In describing and claiming the invention, the following terminology
will be used in accordance with the definitions set forth below.

[0063] The articles "a" and "an" are used herein to refer to one or to
more than one (i.e., to at least one) of the grammatical object of the
article. By way of example, "an element" means one element or more than
one element.

[0064] The term "about" as used herein means greater or lesser than the
value or range of values stated by 10 percent, but is not intended to
designate any value or range of values to only this broader definition.

[0065] A disease or disorder is "alleviated" if the severity of a symptom
of the disease, condition, or disorder, or the frequency with which such
a symptom is experienced by a subject, or both, are reduced.

[0066] As used herein, the term "subject" refers to an individual (e.g.,
human, animal, or other organism) to be treated by the methods or
compositions of the present invention. Subjects include, but are not
limited to, mammals (e.g., murines, simians, equines, bovines, porcines,
canines, felines, and the like), and includes humans. In the context of
the invention, the term "subject" generally refers to an individual who
will receive or who has received treatment for a condition characterized
by the presence of bacteria (e.g., Bacillus anthracis (e.g., in any stage
of its growth cycle), or in anticipation of possible exposure to
bacteria. As used herein, the terms "subject" and "patient" are used
interchangeably, unless otherwise noted.

[0067] As used herein, the terms "neutralize" and "neutralization" when
used in reference to bacterial cells or spores (e.g. B. anthracis cells
and spores) refers to a reduction in the ability of the spores to
germinate and/or cells to proliferate.

[0068] As used herein the term "bacterial spore" or "spore" is used to
refer to any dormant, non-reproductive structure produced by some
bacteria (e.g., Bacillus and Clostridium) in response to adverse
environmental conditions.

[0069] As used herein, the term "treating a surface" refers to the act of
exposing a surface to one or more compositions of the present invention.
Methods of treating a surface include, but are not limited to, spraying,
misting, submerging, wiping, and coating. Surfaces include organic
surfaces (e.g., food products, surfaces of animals, skin, etc.) and
inorganic surfaces (e.g., medical devices, countertops, instruments,
articles of commerce, clothing, etc.).

[0070] As used herein, the term "therapeutically effective amount" refers
to the amount that provides a therapeutic effect, e.g., an amount of a
composition that is effective to treat or prevent pathological
conditions, including signs and/or symptoms of disease, associated with a
pathogenic organism infection (e.g., germination, growth, toxin
production, etc.) in a subject.

[0071] The terms "bacteria" and "bacterium" refer to all prokaryotic
organisms, including those within all of the phyla in the Kingdom
Procaryotae. As used herein, the term "microorganism" refers to any
species or type of microorganism, including but not limited to, bacteria,
archaea, fungi, protozoans, mycoplasma, and parasitic organisms.

[0072] As used herein the term "colonization" refers to the presence of
bacteria in a subject that are either not found in healthy subjects, or
the presence of an abnormal quantity and/or location of bacteria in a
subject relative to a healthy patient.

[0073] The term "stationary growth phase" as used herein defines the
growth characteristics of a given population of microorganisms. During a
stationary growth phase the population of bacteria remains stable with
the rate of bacterial division being approximately equal to the rate of
bacterial death. This may be due to increased generation time of the
bacteria. Accordingly "stationary phase bacteria" are bacteria that are
in a stationary growth phase. "Exponential phase bacteria" are bacteria
that are rapidly proliferating at a rate wherein the population
approximately doubles with each round of division. When the growth rate
(number of cells vs. time) of exponential phase bacteria is graphed, the
plotted data produces an exponential or logarithmic curve.

[0074] As used herein a "multi-drug resistant" microorganism or bacteria
is an organism that has an enhanced ability, relative to non-resistant
strains, to resist distinct drugs or chemicals (of a wide variety of
structure and function) targeted at eradicating the organism. Typically
the term refers to resistance to at least 3 classes of antibiotics.

[0075] Chemokines are small proteins secreted by cells that have the
ability to induce directed chemotaxis in responsive cells. As used herein
the term "interferon-inducible (ELR-) CXC chemokine" refers to a
chemokine protein, or corresponding peptidomimetic, having a motif of
four conserved cysteine residues, the first two of which are separated by
a non-conserved amino acid (thus constituting the Cys-X-Cys or `CXC`
motif; see FIG. 1) and devoid of a three amino acid sequence, glutamic
acid-leucine-arginine (the `ELR` motif), immediately proximal to the CXC
sequence. Examples of interferon-inducible (ELR-)CXC chemokines include
human CXCL9 (SEQ ID NO: 1), murine CXCL9 (SEQ ID NO: 2), human CXCL10
(SEQ ID NO: 4), murine CXCL10 (SEQ ID NO: 5), human CXCL11 (SEQ ID NO: 7)
and murine CXCL11 (SEQ ID NO: 8). CXCL9, CXCL10 and CXCL11 are potently
induced by both type 1 and type 2 interferons (IFN-α/β and
IFN-γ, respectively).

[0076] As used herein the term "lipid vesicle" refers to any spherical
shaped structure formed from amphipathic lipids that surround and enclose
an interior space. The term lipid vesicle encompasses both micelles as
well as liposomes. A micelle is an aggregate of amphipathic lipids with
the hydrophilic "head" regions in contact with surrounding solvent,
sequestering the hydrophobic tail regions in the micelle centre. A
liposome as used herein refers to lipid vesicles comprised of one or more
concentrically ordered lipid bilayers encapsulating an aqueous phase.
Suitable vesicle-forming lipids may be selected from a variety of
amphiphatic lipids, typically including phospho lipids such as
phosphatidylcho line (PC) and, sphingo lipids such as sphingomyelin.

[0077] As used herein, the term "adjuvant" as used herein refers to an
agent which enhances the pharmaceutical effect of another agent.

[0078] As used herein, "amino acids" are represented by the full name
thereof, by the three letter code corresponding thereto, or by the
one-letter code corresponding thereto, as indicated in the following
table:

[0079] The expression "amino acid" as used herein is meant to include both
natural and synthetic amino acids, and both D and L amino acids.
"Standard amino acid" means any of the twenty standard L-amino acids
commonly found in naturally occurring peptides. "Nonstandard amino acid
residue" means any amino acid, other than the standard amino acids,
regardless of whether it is prepared synthetically or derived from a
natural source. As used herein, "synthetic amino acid" also encompasses
chemically modified amino acids, including but not limited to salts,
amino acid derivatives (such as amides), and substitutions. Amino acids
contained within the peptides of the present invention, and particularly
at the carboxy- or amino-terminus, can be modified by methylation,
amidation, acetylation or substitution with other chemical groups which
can change the peptide's circulating half-life without adversely
affecting their activity. Additionally, a disulfide linkage may be
present or absent in the peptides of the invention.

[0080] The term "amino acid" is used interchangeably with "amino acid
residue," and may refer to a free amino acid and to an amino acid residue
of a peptide. It will be apparent from the context in which the term is
used whether it refers to a free amino acid or a residue of a peptide.

[0081] Amino acids have the following general structure:

##STR00001##

[0082] Amino acids may be classified into seven groups on the basis of the
side chain R: (1) aliphatic side chains; (2) side chains containing a
hydroxylic (OH) group; (3) side chains containing sulfur atoms; (4) side
chains containing an acidic or amide group; (5) side chains containing a
basic group; (6) side chains containing an aromatic ring; and (7)
proline, an imino acid in which the side chain is fused to the amino
group.

[0083] As used herein, the term "conservative amino acid substitution" is
defined herein as exchanges within one of the following five groups:

[0092] The nomenclature used to describe the peptide compounds of the
present invention follows the conventional practice wherein the amino
group is presented to the left and the carboxy group to the right of each
amino acid residue. In the formulae representing selected specific
embodiments of the present invention, the amino-and carboxy-terminal
groups, although not specifically shown, will be understood to be in the
form they would assume at physiologic pH values, unless otherwise
specified.

[0093] The term "basic" or "positively charged" amino acid, as used
herein, refers to amino acids in which the R groups have a net positive
charge at pH 7.0, and include, but are not limited to, the standard amino
acids lysine, arginine, and histidine.

[0094] As used herein, an "analog" of a chemical compound is a compound
that, by way of example, resembles another in structure but is not
necessarily an isomer (e.g., 5-fluorouracil is an analog of thymine).

[0095] The term "antibody," as used herein, refers to an immunoglobulin
molecule which is able to specifically bind to a specific epitope on an
antigen. Antibodies can be derived from natural sources or from
recombinant sources and may be intact immunoglobulins, or immunoreactive
portions of intact immunoglobulins. Antibodies are typically tetramers of
immunoglobulin molecules. The antibodies in the present invention may
exist in a variety of forms including, for example, polyclonal
antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well as
single chain antibodies and humanized antibodies (Harlow et al., 1999,
Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory
Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold
Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA
85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0096] By the term "synthetic antibody" as used herein, is meant an
antibody which is generated using recombinant DNA technology, such as,
for example, an antibody expressed by a bacteriophage as described
herein. The term should also be construed to mean an antibody which has
been generated by the synthesis of a DNA molecule encoding the antibody
and which DNA molecule expresses an antibody protein, or an amino acid
sequence specifying the antibody, wherein the DNA or amino acid sequence
has been obtained using synthetic DNA or amino acid sequence technology
which is available and well known in the art.

[0097] The term "antimicrobial agent", as used herein, refers to any
entity that exhibits antimicrobial activity, i.e. the ability to inhibit
the growth and/or kill bacteria, including for example the ability to
inhibit growth or reduce viability of bacteria by at least about 30%, at
least about 40%, at least about 50%, at least about 60%, at least about
70% or more than 70%, as compared to bacteria not exposed to the
antimicrobial agent. The antimicrobial agent can exert its effect either
directly or indirectly and can be selected from a library of diverse
compounds, including for example antibiotics. For example, various
antimicrobial agents act, inter alia, by interfering with (1) cell wall
synthesis, (2) plasma membrane integrity, (3) nucleic acid synthesis, (4)
ribosomal function, and (5) folate synthesis. One of ordinary skill in
the art will appreciate that a number of "antimicrobial susceptibility"
tests can be used to determine the efficacy of a candidate antimicrobial
agent.

[0098] As used herein, the term "antisense oligonucleotide" means a
nucleic acid polymer, at least a portion of which is complementary to a
nucleic acid which is present in a normal cell or in an affected cell.
The antisense oligonucleotides of the invention include, but are not
limited to, phosphorothioate oligonucleotides and other modifications of
oligonucleotides. Methods for synthesizing oligonucleotides,
phosphorothioate oligonucleotides, and otherwise modified
oligonucleotides are well known in the art (U.S. Pat. No: 5,034,506;
Nielsen et al., 1991, Science 254: 1497). "Antisense" refers particularly
to the nucleic acid sequence of the non-coding strand of a double
stranded DNA molecule encoding a protein, or to a sequence which is
substantially homologous to the non-coding strand. As defined herein, an
antisense sequence is complementary to the sequence of a double stranded
DNA molecule encoding a protein. It is not necessary that the antisense
sequence be complementary solely to the coding portion of the coding
strand of the DNA molecule. The antisense sequence may be complementary
to regulatory sequences specified on the coding strand of a DNA molecule
encoding a protein, which regulatory sequences control expression of the
coding sequences.

[0099] As used herein, the term "biologically active fragments" or "bio
active fragment" of the polypeptides encompasses natural or synthetic
portions of the full-length protein that are capable of specific binding
to their natural ligand or of performing the function of the protein.

[0100] A "pathogenic" cell is a cell which, when present in a tissue,
causes or contributes to a disease or disorder in the animal in which the
tissue is located (or from which the tissue was obtained).

[0101] "Complementary" refers to the broad concept of sequence
complementarity between regions of two nucleic acid strands or between
two regions of the same nucleic acid strand. It is known that an adenine
residue of a first nucleic acid region is capable of forming specific
hydrogen bonds ("base pairing") with a residue of a second nucleic acid
region which is antiparallel to the first region if the residue is
thymine or uracil. As used herein, the terms "complementary" or
"complementarity" are used in reference to polynucleotides (i.e., a
sequence of nucleotides) related by the base-pairing rules. For example,
for the sequence "A-G-T," is complementary to the sequence "T-C-A."

[0102] Similarly, it is known that a cytosine residue of a first nucleic
acid strand is capable of base pairing with a residue of a second nucleic
acid strand which is antiparallel to the first strand if the residue is
guanine A first region of a nucleic acid is complementary to a second
region of the same or a different nucleic acid if, when the two regions
are arranged in an antiparallel fashion, at least one nucleotide residue
of the first region is capable of base pairing with a residue of the
second region. Preferably, the first region comprises a first portion and
the second region comprises a second portion, whereby, when the first and
second portions are arranged in an antiparallel fashion, at least about
50%, and preferably at least about 75%, at least about 90%, or at least
about 95% of the nucleotide residues of the first portion are capable of
base pairing with nucleotide residues in the second portion. More
preferably, all nucleotide residues of the first portion are capable of
base pairing with nucleotide residues in the second portion.

[0103] The terms "cell" and "cell line," as used herein, may be used
interchangeably. All of these terms also include their progeny, which are
any and all subsequent generations. It is understood that all progeny may
not be identical due to deliberate or inadvertent mutations.

[0104] The terms "cell culture" and "culture," as used herein, refer to
the maintenance of cells in an artificial, in vitro environment. It is to
be understood, however, that the term "cell culture" is a generic term
and may be used to encompass the cultivation not only of individual
cells, but also of tissues, organs, organ systems or whole organisms, for
which the terms "tissue culture," "organ culture," "organ system culture"
or "organotypic culture" may occasionally be used interchangeably with
the term "cell culture."

[0105] The phrases "cell culture medium," "culture medium" (plural "media"
in each case) and "medium formulation" refer to a nutritive solution for
cultivating cells and may be used interchangeably.

[0106] A "conditioned medium" is one prepared by culturing a first
population of cells or tissue in a medium, and then harvesting the
medium. The conditioned medium (along with anything secreted into the
medium by the cells) may then be used to support the growth or
differentiation of a second population of cells.

[0107] The term "complex", as used herein in reference to proteins, refers
to binding or interaction of two or more proteins. Complex formation or
interaction can include such things as binding, changes in tertiary
structure, and modification of one protein by another, such as
phosphorylation.

[0108] A "compound", as used herein, refers to any type of substance or
agent that is commonly considered a chemical, drug, or a candidate for
use as a drug, as well as combinations and mixtures of the above. The
term compound further encompasses molecules such as peptides and nucleic
acids.

[0109] "Cytokine," as used herein, refers to intercellular signaling
molecules, the best known of which are involved in the regulation of
mammalian somatic cells. A number of families of cytokines, both growth
promoting and growth inhibitory in their effects, have been characterized
including, for example, interleukins, interferons, and transforming
growth factors. A number of other cytokines are known to those of skill
in the art. The sources, characteristics, targets and effector activities
of these cytokines have been described.

[0110] As used herein, a "derivative" of a compound refers to a chemical
compound that may be produced from another compound of similar structure
in one or more steps, as in replacement of H by an alkyl, acyl, or amino
group.

[0111] As used herein, a "detectable marker" or a "reporter molecule" is
an atom or a molecule that permits the specific detection of a compound
comprising the marker in the presence of similar compounds without a
marker. Detectable markers or reporter molecules include, e.g.,
radioactive isotopes, antigenic determinants, enzymes, nucleic acids
available for hybridization, chromophores, fluorophores, chemiluminescent
molecules, electrochemically detectable molecules, and molecules that
provide for altered fluorescence-polarization or altered
light-scattering.

[0112] A "disease" is a state of health of an animal wherein the animal
cannot maintain homeostasis, and wherein if the disease is not
ameliorated then the animal's health continues to deteriorate.

[0113] In contrast, a "disorder" in an animal is a state of health in
which the animal is able to maintain homeostasis, but in which the
animal's state of health is less favorable than it would be in the
absence of the disorder. Left untreated, a disorder does not necessarily
cause a further decrease in the animal's state of health.

[0114] "Encoding" refers to the inherent property of specific sequences of
nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to
serve as templates for synthesis of other polymers and macromolecules in
biological processes having either a defined sequence of nucleotides
(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the
biological properties resulting therefrom. Thus, a gene encodes a protein
if transcription and translation of mRNA corresponding to that gene
produces the protein in a cell or other biological system. Both the
coding strand, the nucleotide sequence of which is identical to the mRNA
sequence and is usually provided in sequence listings, and the non-coding
strand, used as the template for transcription of a gene or cDNA, can be
referred to as encoding the protein or other product of that gene or
cDNA.

[0115] Unless otherwise specified, a "nucleotide sequence encoding an
amino acid sequence" includes all nucleotide sequences that are
degenerate versions of each other and that encode the same amino acid
sequence. Nucleotide sequences that encode proteins and RNA may include
introns.

[0116] As used herein, an "essentially pure" preparation of a particular
protein or peptide is a preparation wherein at least about 95%, and
preferably at least about 99%, by weight, of the protein or peptide in
the preparation is the particular protein or peptide.

[0117] A "fragment" or "segment" is a portion of an amino acid sequence,
comprising at least one amino acid, or a portion of a nucleic acid
sequence comprising at least one nucleotide. The terms "fragment" and
"segment" are used interchangeably herein.

[0118] As used herein, a "functional" biological molecule is a biological
molecule in a form in which it exhibits a property or activity by which
it is characterized. A functional enzyme, for example, is one which
exhibits the characteristic catalytic activity by which the enzyme is
characterized.

[0119] The terms "formula" and "structure" are used interchangeably
herein.

[0120] The term "identity" as used herein relates to the similarity
between two or more sequences. Identity is measured by dividing the
number of identical residues by the total number of residues and
multiplying the product by 100 to achieve a percentage. Thus, two copies
of exactly the same sequence have 100% identity, whereas two sequences
that have amino acid deletions, additions, or substitutions relative to
one another have a lower degree of identity. Those skilled in the art
will recognize that several computer programs, such as those that employ
algorithms such as BLAST (Basic Local Alignment Search Tool, Altschul et
al. (1993) J. Mol. Biol. 215:403-410) are available for determining
sequence identity.

[0121] The term "inhibit," as used herein, refers to the ability of a
compound or any agent to reduce or impede a described function or
pathway. For example, inhibition can be by at least 10%, by at least 25%,
by at least 50%, and even by at least 75%.

[0122] As used herein, an "instructional material" includes a publication,
a recording, a diagram, or any other medium of expression which can be
used to communicate the usefulness of the peptide of the invention in the
kit for effecting alleviation of the various diseases or disorders
recited herein. Optionally, or alternately, the instructional material
may describe one or more methods of alleviating the diseases or disorders
in a cell or a tissue of a mammal. The instructional material of the kit
of the invention may, for example, be affixed to a container which
contains the identified compound invention or be shipped together with a
container which contains the identified compound. Alternatively, the
instructional material may be shipped separately from the container with
the intention that the instructional material and the compound be used
cooperatively by the recipient.

[0123] An "isolated" compound/moiety is a compound/moeity that has been
removed from components naturally associated with the compound/moiety.
For example, an "isolated nucleic acid" refers to a nucleic acid segment
or fragment which has been separated from sequences which flank it in a
naturally occurring state, e.g., a DNA fragment which has been removed
from the sequences which are normally adjacent to the fragment, e.g., the
sequences adjacent to the fragment in a genome in which it naturally
occurs. The term also applies to nucleic acids which have been
substantially purified from other components which naturally accompany
the nucleic acid, e.g., RNA or DNA or proteins, which naturally accompany
it in the cell. The term therefore includes, for example, a recombinant
DNA which is incorporated into a vector, into an autonomously replicating
plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote,
or which exists as a separate molecule (e.g., as a cDNA or a genomic or
cDNA fragment produced by PCR or restriction enzyme digestion)
independent of other sequences. It also includes a recombinant DNA which
is part of a hybrid gene encoding additional polypeptide sequence.

[0124] The term "modulate", as used herein, refers to changing the level
of an activity, function, or process. The term "modulate" encompasses
both inhibiting and stimulating an activity, function, or process.

[0125] Unless otherwise specified, a "nucleotide sequence encoding an
amino acid sequence" includes all nucleotide sequences that are
degenerate versions of each other and that encode the same amino acid
sequence. Nucleotide sequences that encode proteins and RNA may include
introns.

[0126] The term "Oligonucleotide" typically refers to short
polynucleotides, generally no greater than about 50 nucleotides. It will
be understood that when a nucleotide sequence is represented by a DNA
sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A,
U, G, C) in which "U" replaces "T."

[0127] As used herein, the term "purified" and like terms relate to an
enrichment of a molecule or compound relative to other components
normally associated with the molecule or compound in a native
environment. The term "purified" does not necessarily indicate that
complete purity of the particular molecule has been achieved during the
process. A "highly purified" compound as used herein refers to a compound
that is greater than 90% pure.

[0128] As used herein, the term "pharmaceutically acceptable carrier"
includes any of the standard pharmaceutical carriers, such as a phosphate
buffered saline solution, water, emulsions such as an oil/water or
water/oil emulsion, and various types of wetting agents. The term also
encompasses any of the agents approved by a regulatory agency of the US
Federal government or listed in the US Pharmacopeia for use in animals,
including humans.

[0132] A "recombinant polypeptide" is one which is produced upon
expression of a recombinant polynucleotide.

[0133] A peptide encompasses a sequence of 2 or more amino acids wherein
the amino acids are naturally occurring or synthetic (non-naturally
occurring) amino acids.

[0134] The term "linked" or like terms refers to a connection between two
entities. The linkage may comprise a covalent, ionic, or hydrogen bond or
other interaction that binds two compounds or substances to one another.

[0135] As used herein the term "peptidomimetic" refers to a chemical
compound having a structure that is different from the general structure
of an existing peptide, but that functions in a manner similar to the
existing peptide, e.g., by mimicking the biological activity of that
peptide. Peptidomimetics typically comprise naturally-occurring amino
acids and/or unnatural amino acids, but can also comprise modifications
to the peptide backbone. For example a peptidomimetic may include one or
more of the following modifications:

[0136] 1. peptides wherein one or more of the peptidyl --C(O)NR-- linkages
(bonds) have been replaced by a non-peptidyl linkage such as a
--CH2-carbamate linkage (--CH2OC(O)NR--), a phosphonate linkage, a
--CH2-sulfonamide (--CH2--S(O)2NR--) linkage, a urea (--NHC(O)NH--)
linkage, a --CH2-secondary amine linkage, an azapeptide bond (CO
substituted by NH), or an ester bond (e.g., depsipeptides, wherein one or
more of the amide (--CONHR--) bonds are replaced by ester (COOR) bonds)
or with an alkylated peptidyl linkage (--C(O)NR--) wherein R is C1-C4
alkyl;

[0137] 2. peptides wherein the N-terminus is derivatized to a --NRR1
group, to a --NRC(O)R group, to a --NRC(O)OR group, to a --NRS(O)2R
group, to a --NHC(O)NHR group where R and R1 are hydrogen or C1-C4 alkyl
with the proviso that R and R1 are not both hydrogen;

[0138] 3. peptides wherein the C terminus is derivatized to --C(O)R2 where
R2 is selected from the group consisting of C1-C4 alkoxy, and --NR3R4
where R3 and R4 are independently selected from the group consisting of
hydrogen and C1-C4 alkyl;

[0139] 4. modification of a sequence of naturally-occurring amino acids
with the insertion or substitution of a non-peptide moiety, e.g. a
retroinverso fragment.

[0140] The term "permeability," as used herein, refers to transit of
fluid, cell, or debris between or through cells and tissues.

[0141] As used herein, the term "pharmaceutically acceptable carrier"
includes any of the standard pharmaceutical carriers, such as a phosphate
buffered saline solution, water, emulsions such as an oil/water or
water/oil emulsion, and various types of wetting agents. The term also
encompasses any of the agents approved by a regulatory agency of the US
Federal government or listed in the US Pharmacopeia for use in animals,
including humans.

[0142] As used herein, "protecting group" with respect to a terminal amino
group refers to a terminal amino group of a peptide, which terminal amino
group is coupled with any of various amino-terminal protecting groups
traditionally employed in peptide synthesis. Such protecting groups
include, for example, acyl protecting groups such as formyl, acetyl,
benzoyl, trifluoroacetyl, succinyl, and methoxysuccinyl; aromatic
urethane protecting groups such as benzyloxycarbonyl; and aliphatic
urethane protecting groups, for example, tert-butoxycarbonyl or
adamantyloxycarbonyl. See Gross and Mienhofer, eds., The Peptides, vol.
3, pp. 3-88 (Academic Press, New York, 1981) for suitable protecting
groups.

[0143] As used herein, "protecting group" with respect to a terminal
carboxy group refers to a terminal carboxyl group of a peptide, which
terminal carboxyl group is coupled with any of various carboxyl-terminal
protecting groups. Such protecting groups include, for example,
tert-butyl, benzyl or other acceptable groups linked to the terminal
carboxyl group through an ester or ether bond.

[0144] A "sample," as used herein, refers preferably to a biological
sample from a subject, including, but not limited to, normal tissue
samples, diseased tissue samples, biopsies, blood, saliva, feces, semen,
tears, and urine. A sample can also be any other source of material
obtained from a subject which contains cells, tissues, or fluid of
interest. A sample can also be obtained from cell or tissue culture.

[0145] By the term "specifically binds," as used herein, is meant a
compound which recognizes and binds a specific protein, but does not
substantially recognize or bind other molecules in a sample, or it means
binding between two or more proteins as in part of a cellular regulatory
process, where said proteins do not substantially recognize or bind other
proteins in a sample. The term "standard," as used herein, refers to
something used for comparison. For example, it can be a known standard
agent or compound which is administered or added to a control sample and
used for comparing results when measuring said compound in a test sample.
Standard can also refer to an "internal standard", such as an agent or
compound which is added at known amounts to a sample and is useful in
determining such things as purification or recovery rates when a sample
is processed or subjected to purification or extraction procedures before
a marker of interest is measured.

[0146] The term "symptom," as used herein, refers to any morbid phenomenon
or departure from the normal in structure, function, or sensation,
experienced by the patient and indicative of disease. In contrast, a sign
is objective evidence of disease. For example, a bloody nose is a sign.
It is evident to the patient, doctor, nurse and other observers.

[0147] As used herein, the term "treating" includes prophylaxis of the
specific disorder or condition, or alleviation of the symptoms associated
with a specific disorder or condition and/or preventing or eliminating
said symptoms. A "prophylactic" treatment is a treatment administered to
a subject who does not exhibit signs of a disease or exhibits only early
signs of the disease for the purpose of decreasing the risk of developing
pathology associated with the disease.

[0148] A "therapeutic" treatment is a treatment administered to a subject
who exhibits signs of pathology for the purpose of diminishing or
eliminating those signs.

[0149] As used herein an "amino acid modification" refers to a
substitution, addition or deletion of an amino acid, and includes
substitution with, or addition of, any of the 20 amino acids commonly
found in human proteins, as well as unusual or non-naturally occurring
amino acids. Commercial sources of unusual amino acids include
Sigma-Aldrich (Milwaukee, Wis.), ChemPep Inc. (Miami, Fla.), and Genzyme
Pharmaceuticals (Cambridge, Mass.). Unusual amino acids may be purchased
from commercial suppliers, synthesized de novo, or chemically modified or
derivatized from naturally occurring amino acids. Amino acid
modifications include linkage of an amino acid to a conjugate moiety,
such as a hydrophilic polymer, acylation, alkylation, and/or other
chemical derivatization of an amino acid.

[0150] Modifications (which do not normally alter primary sequence)
include in vivo, or in vitro chemical derivatization of polypeptides,
e.g., acetylation, or carboxylation. Also included are modifications of
glycosylation, e.g., those made by modifying the glycosylation patterns
of a polypeptide during its synthesis and processing or in further
processing steps; e.g., by exposing the polypeptide to enzymes which
affect glycosylation, e.g., mammalian glycosylating or deglycosylating
enzymes. Also embraced are sequences which have phosphorylated amino acid
residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine.

[0151] Also included are polypeptides which have been modified using
ordinary molecular biological techniques so as to improve their
resistance to proteolytic degradation or to optimize solubility
properties or to render them more suitable as a therapeutic agent.
Analogs of such polypeptides include those containing residues other than
naturally occurring L-amino acids, e.g., D-amino acids or non-naturally
occurring synthetic amino acids. The peptides of the invention are not
limited to products of any of the specific exemplary processes listed
herein.

[0152] Substitutions may be designed based on, for example, the model of
Dayhoff, et al. (in Atlas of Protein Sequence and Structure 1978, Nat'l
Biomed. Res. Found., Washington D.C.).

[0153] Conservative substitutions are likely to be phenotypically silent.
Typically seen as conservative substitutions are the replacements, one
for another, among the aliphatic amino acids Ala, Val, Leu, and Ile;
interchange of the hydroxyl residues Ser and Thr, exchange of the acidic
residues Asp and Glu, substitution between the amide residues Asn and
Gln, exchange of the basic residues Lys and Arg and replacements among
the aromatic residues Phe, Tyr. Guidance concerning which amino acid
changes are likely to be phenotypically silent are found in Bowie et al.,
Science 247:1306-1310 (1990).

[0154] The peptides of the present invention may be readily prepared by
standard, well-established techniques, such as solid-phase peptide
synthesis (SPPS) as described by Stewart et al. in Solid Phase Peptide
Synthesis, 2nd Edition, 1984, Pierce Chemical Company, Rockford, Ill.;
and as described by Bodanszky and Bodanszky in The Practice of Peptide
Synthesis, 1984, Springer-Verlag, New York. At the outset, a suitably
protected amino acid residue is attached through its carboxyl group to a
derivatized, insoluble polymeric support, such as cross-linked
polystyrene or polyamide resin. "Suitably protected" refers to the
presence of protecting groups on both the α-amino group of the
amino acid, and on any side chain functional groups. Side chain
protecting groups are generally stable to the solvents, reagents and
reaction conditions used throughout the synthesis, and are removable
under conditions which will not affect the final peptide product.
Stepwise synthesis of the oligopeptide is carried out by the removal of
the N-protecting group from the initial amino acid, and couple thereto of
the carboxyl end of the next amino acid in the sequence of the desired
peptide. This amino acid is also suitably protected. The carboxyl of the
incoming amino acid can be activated to react with the N-terminus of the
support-bound amino acid by formation into a reactive group such as
formation into a carbodiimide, a symmetric acid anhydride, or an "active
ester" group such as hydroxybenzotriazole or pentafluorophenly esters.

[0155] Examples of solid phase peptide synthesis methods include the BOC
method which utilized tert-butyloxcarbonyl as the α-amino
protecting group, and the FMOC method which utilizes
9-fluorenylmethyloxcarbonyl to protect the a-amino of the amino acid
residues, both methods of which are well-known by those of skill in the
art.

[0156] Incorporation of N- and/or C-blocking groups can also be achieved
using protocols conventional to solid phase peptide synthesis methods.
For incorporation of C-terminal blocking groups, for example, synthesis
of the desired peptide is typically performed using, as solid phase, a
supporting resin that has been chemically modified so that cleavage from
the resin results in a peptide having the desired C-terminal blocking
group. To provide peptides in which the C-terminus bears a primary amino
blocking group, for instance, synthesis is performed using a
p-methylbenzhydrylamine (MBHA) resin so that, when peptide synthesis is
completed, treatment with hydrofluoric acid releases the desired
C-terminally amidated peptide. Similarly, incorporation of an
N-methylamine blocking group at the C-terminus is achieved using
N-methylaminoethyl-derivatized DVB, resin, which upon HF treatment
releases a peptide bearing an N-methylamidated C-terminus. Blockage of
the C-terminus by esterification can also be achieved using conventional
procedures. This entails use of resin/blocking group combination that
permits release of side-chain peptide from the resin, to allow for
subsequent reaction with the desired alcohol, to form the ester function.
FMOC protecting group, in combination with DVB resin derivatized with
methoxyalkoxybenzyl alcohol or equivalent linker, can be used for this
purpose, with cleavage from the support being effected by TFA in
dicholoromethane. Esterification of the suitably activated carboxyl
function e.g. with DCC, can then proceed by addition of the desired
alcohol, followed by deprotection and isolation of the esterified peptide
product.

[0157] Incorporation of N-terminal blocking groups can be achieved while
the synthesized peptide is still attached to the resin, for instance by
treatment with a suitable anhydride and nitrile. To incorporate an acetyl
blocking group at the N-terminus, for instance, the resincoupled peptide
can be treated with 20% acetic anhydride in acetonitrile. The N-blocked
peptide product can then be cleaved from the resin, deprotected and
subsequently isolated.

[0158] To ensure that the peptide obtained from either chemical or
biological synthetic techniques is the desired peptide, analysis of the
peptide composition should be conducted. Such amino acid composition
analysis may be conducted using high resolution mass spectrometry to
determine the molecular weight of the peptide. Alternatively, or
additionally, the amino acid content of the peptide can be confirmed by
hydrolyzing the peptide in aqueous acid, and separating, identifying and
quantifying the components of the mixture using HPLC, or an amino acid
analyzer. Protein sequenators, which sequentially degrade the peptide and
identify the amino acids in order, may also be used to determine
definitely the sequence of the peptide.

[0159] Prior to its use, the peptide is purified to remove contaminants.
In this regard, it will be appreciated that the peptide will be purified
so as to meet the standards set out by the appropriate regulatory
agencies. Any one of a number of a conventional purification procedures
may be used to attain the required level of purity including, for
example, reversed-phase high-pressure liquid chromatography (HPLC) using
an alkylated silica column such as C4-, C8- or C18-silica.
A gradient mobile phase of increasing organic content is generally used
to achieve purification, for example, acetonitrile in an aqueous buffer,
usually containing a small amount of trifluoroacetic acid. Ion-exchange
chromatography can be also used to separate peptides based on their
charge.

[0160] Substantially pure protein obtained as described herein may be
purified by following known procedures for protein purification, wherein
an immunological, enzymatic or other assay is used to monitor
purification at each stage in the procedure. Protein purification methods
are well known in the art, and are described, for example in Deutscher et
al. (ed., 1990, Guide to Protein Purification, Harcourt Brace Jovanovich,
San Diego).

Embodiments

[0161] In accordance with one embodiment compositions and methods are
provided for neutralizing pathogenic organisms. More particularly,
applicants have found that interferon-inducible ELR- CXC chemokines have
efficacy against pathogenic bacteria including Bacillus anthraci. In
accordance with one embodiment a composition is provided for neutralizing
pathogenic bacteria in all growth phases including sporulated forms. The
compositions can be formulated for treatment of external surfaces
including hard surfaces such as, medical equipment and medical devices,
or the compositions can be formulated for topical or internal
administration to subjects, including humans.

[0162] In accordance with one embodiment a composition is provided
comprising a non-native peptide, or a peptidomimetic derivative,
comprising a sequence selected from the group consisting of SEQ ID NO: 3,
SEQ ID NO: 6 and SEQ ID NO: 9 or a sequence that differs from SEQ ID NO:
3, SEQ ID NO: 6 or SEQ ID NO: 9 by 1, 2, 3, 4 or 5 amino acids. In one
embodiment the peptide differs from SEQ ID NO: 3, SEQ ID NO: 6 or SEQ ID
NO: 9 by 1, 2, 3, 4 or 5 conservative amino acid substitutions. In
accordance with one embodiment a composition is provided comprising a
peptide, or a peptidomimetic derivative, comprising a sequence selected
from the group consisting of SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 9.
In a further embodiment a composition is provided comprising a non-native
peptide, or a peptidomimetic derivative thereof, comprising a sequence
selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 13 or SEQ
ID NO: 16.

[0163] In another embodiment the non-native peptide, or peptidomimetic
derivative thereof, comprises a sequence selected from the group
consisting of SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14 or a
sequence that differs from SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14
by 1, 2, 3, 4 or 5 amino acids. In another embodiment the peptide, or
peptidomimetic derivative, comprises a sequence selected from the group
consisting of SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17 or a
sequence that differs from SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17
by 1, 2, 3, 4 or 5 amino acids. In another embodiment a composition is
provided comprising a non-native peptide, or a peptidomimetic derivative,
comprising a sequence selected from the group consisting of SEQ ID NO: 1,
SEQ ID NO: 4 or SEQ ID NO: 7, and in further embodiment the sequence
comprises the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 16, or a
peptidomimetic derivative thereof.

[0166] In some embodiments, the peptide of the present disclosures
comprises an amino acid sequence which has at least 95% sequence identity
to an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 7.
In some embodiments, the peptide of the present disclosures comprises an
amino acid sequence which is at least 70%, at least 80%, at least 85%, at
least 90% or has greater than 95% sequence identity to SEQ ID NO: 4. In
some embodiments, the amino acid sequence of the presently disclosed
peptide which has the above-referenced % sequence identity is the
full-length amino acid sequence of the presently disclosed peptide.

[0167] In one embodiment an antimicrobial composition is provided
comprising two or more interferon-inducible (ELR-) CXC chemokines. In one
embodiment the composition comprises a purified first peptide having the
sequence of SEQ ID NO; 12 or SEQ ID NO: 15, and a purified second peptide
having the sequence of SEQ ID NO: 13 or SEQ ID NO: 16. In one embodiment
the composition comprises a non-native first peptide having the sequence
of SEQ ID NO: 15, and a non-native second peptide having the sequence of
SEQ ID NO: 16.

[0168] It is further contemplated that the antimicrobial
interferon-inducible (ELR-) CXC chemokines disclosed herein may be used
in combination with, or to enhance the activity of, other antimicrobial
agents or antibiotics. In one embodiment a composition is provided
comprising an interferon-inducible (ELR-) CXC chemokine and a second
antimicrobial agent. In one embodiment the second antimicrobial agent is
an antibiotic. Combinations of an interferon-inducible (ELR-) CXC
chemokine peptide (or other compounds identified by the methods disclosed
herein) with other agents may be useful to allow antibiotics to be used
at lower doses responsive to toxicity concerns, to enhance the activity
of antibiotics whose efficacy has been reduced or to effectuate a
synergism between the components such that the combination is more
effective than the sum of the efficacy of either component independently.

[0169] In some embodiments, the antimicrobial agent is a quinolone
antimicrobial agent, including for example but not limited to,
ciprofloxacin, levofloxacin, and ofloxacin, gatifloxacin, norfloxacin,
lomefloxacin, trovafloxacin, moxifloxacin, sparfloxacin, gemifloxacin,
pazufloxacin or variants or analogues thereof. In some embodiments, the
second antimicrobial agent is ofloxacin or variants or analogues thereof.

[0170] In some embodiments, the second antimicrobial agent is an
aminoglycoside antimicrobial agent, including for example but not limited
to, amikacin, gentamycin, tobramycin, netromycin, streptomycin,
kanamycin, paromomycin, neomycin or variants or analogues thereof In some
embodiments, the second antimicrobial agent is gentamicin or variants or
analogues thereof.

[0171] In some embodiments, the second antimicrobial agent is a
beta-lactam antibiotic antimicrobial agent, including for example but not
limited to, penicillin, ampicillin, penicillin derivatives,
cephalosporins, monobactams, carbapenems, beta-lactamase inhibitors or
variants or analogues thereof In some embodiments, the second
antimicrobial agent is ampicillin or variants or analogues thereof In
accordance with one embodiment the second antimicrobial agent is selected
from a group consisting of penicillin, ampicillin, penicillin
derivatives, cephalosporins, monobactams, carbapenems, or beta-lactamase
inhibitors.

[0172] The compositions disclosed herein may include additional components
that enhance their efficacy based on their desired use. In one embodiment
the compositions are formulated as a pharmaceutical composition. The
pharmaceutical compositions can be prepared for systemic (parenteral),
inhalational (or inhaled), and topical applications using formulations
and techniques known to those skilled in the art. Such pharmaceutical
compositions include one or more isolated or purified
interferon-inducible (ELR-) CXC chemokines, or pharmaceutically
acceptable salts thereof, and a pharmaceutically acceptable carrier.

[0174] In one embodiment the interferon-inducible (ELR-) CXC chemokine may
be coupled, bonded, bound, conjugated, or chemically-linked to one or
more agents via linkers, polylinkers, or derivatized amino acids. In
accordance with one embodiment the composition further comprises a lipid
vesicle delivery vehicle. In one embodiment the lipid vesicle is a
liposome or micelle. Suitable lipids for liposomal and/or micelle
formulation include, without limitation, monoglycerides, diglycerides,
sulfatides, lysolecithin, phospholipid, saponin, bile acids, and the
like. The preparation of liposomal formulations is within the level of
skill in the art, as disclosed, for example, in U.S. Pat. No. 4,235,871;
U.S. Pat. No. 4,501,728; U.S. Pat. No. 4,837,028; and U.S. Pat. No.
4,737,323, the disclosures of which are incorporated herein by reference.
In accordance with one embodiment a composition is provided comprising an
interferon-inducible (ELR-) CXC chemokine and a lipid vesicle, wherein
the interferon-inducible (ELR-) CXC chemokine is encapsulated within the
lipid vesicle, or linked to the surface of said lipid vesicle. In a
further embodiment the composition may include additional active agents
encapsulated or linked to the surface of the lipid vesicle delivery
vehicle, including for example an anti-microbial agent such as an
antibiotic. In one embodiment the lipid vesicle is a liposome, and in a
further embodiment the liposome comprises interferon-inducible (ELR-) CXC
chemokines linked to the exterior surface of the liposome. In one
embodiment the interferon-inducible (ELR-) CXC chemokines are covalently
bound to the exterior surface of the liposome, optionally with additional
active antimicrobial agents encapsulated within or linked to the exterior
surface of the liposome.

[0176] In one embodiment the antibiotics that are combined with the
interferon-inducible (ELR-) CXC chemokine include but are not limited to
penicillin, ampicillin, amoxycillin, vancomycin, cycloserine, bacitracin,
cephalolsporin, methicillin, streptomycin, kanamycin, tobramycin,
gentamicin, tetracycline, chlortetracycline, doxycycline,
chloramphenicol, lincomycin, clindamycin, erythromycin, oleandomycin,
polymyxin nalidixic acid, rifamycin, rifampicin, gantrisin, trimethoprim,
isoniazid, paraminosalicylic acid, and ethambutol. In some embodiments,
the antibiotic comprises one or more anti-anthrax agents (e.g., an
antibiotic used in the art for treating B. anthracis (e.g., penicillin,
ciprofloxacin, doxycycline, erythromycin, and vancomycin)).

[0177] In one embodiment a kit is provided for neutralizing pathogenic
organisms. In one embodiment the kit comprises an interferon-inducible
(ELR-) CXC chemokine (as disclosed herein) and additional known
antimicrobial agents, including one or more antibiotics. In a further
embodiment the kit comprises a type 1 and/or type 2 interferons (e.g.,
IFN-α/β and IFN-γ, respectively).

Neutralizing Stationary Phase Bacteria

[0178] Many antibiotics are only poorly effective against non-growing or
stationary phase bacteria. During the stationary period bacterial cells
frequently have a thicker peptidoglycan cell wall and typically have
differences in metabolism and protein synthesis. Many complications that
arise during the course of treating bacterial infections are due to
stationary phase or dormant bacteria, which as noted above resist
conventional antibiotic treatments. The formation of bio films on
temporary (e.g., catheters) or more permanent implants and the
colonizations seen in patients afflicted with certain diseases cannot be
effectively treated with the antimicrobial agents currently available. In
terms of bacterial colonization and diseases that can arise from it, the
airways and the GI tract are the major areas affected. The presence of
inappropriate bacterial colonizations is believed to cause complications
associated with inflammatory bowel diseases (including ulcerative colitis
and Crohn's disease) and irritable bowel syndrome. In addition with
regards to the airways alone, the major diseases that can arise from, or
can be exacerbated by, bacterial colonization include: sinus infections,
respiratory infections such as pneumonia (this is especially applicable
to ventilator-associated pneumonias but also applies to
community-acquired pneumonias), chronic obstructive pulmonary disease
(COPD), and cystic fibrosis (CF).

[0179] Surprisingly, applicants have discovered that the
interferon-inducible (ELR-) CXC chemokines have activity in neutralizing
stationary phase bacteria as well as actively growing bacteria (see FIGS.
11A-11C). In accordance with one embodiment a method is provided for
neutralizing prokaryotic pathogenic organisms that have colonized a host
organism and have entered into a stationary growth phase. It is also
anticipated that the interferon-inducible (ELR-) CXC chemokine containing
compositions may have efficacy in neutralizing biofilms. The method
comprises the step of contacting the pathogenic organisms with a
composition comprising an interferon-inducible (ELR-) CXC chemokine

[0181] Since interferons are known to induce expression of native CXCL9,
CXCL10 and CXCL11, in one embodiment the method of treatment comprises
the co-administration of one or more interferons, including for example
interferon-alpha, interferon-beta and/or interferon-gamma as an adjuvant
to promote production of native CXCL9, CXCL10 and CXCL11 chemokines in
vivo. Co-aministration can be accomplished by simultaneously
administering the chemokine and the interferon, or the two active agents
can be administered one after the other within 1, 2, 3, 4, 5, 6, 12, 24
or 48 hours of each other.

[0182] The pathogenic organisms are placed in contact using an appropriate
route of administration. For example, for treating skin, the composition
can be formulated as a topical cream, ointment or in a rinsing solution.
Such composition can be used sterilize external body parts that may have
come in contact with pathogenic organisms such as Bacillus anthracis.
Alternatively, formulations for oral administration can be prepared for
treating bacterial colonization of the digestive tract. In another
embodiment the composition can be formulated as an aerosol for
administration to the lungs and air pathways of a subject. Such
formulations can be prepared using standard formulations and techniques
known to the skilled practitioner.

[0183] The interferon-inducible (ELR-) CXC chemokine compositions will be
administered in an amount effective to neutralize the bacteria. An
"effective" amount or a "therapeutically effective amount" of the
interferon-inducible (ELR-) CXC chemokine refers to a nontoxic but
sufficient amount of the compound to provide the desired effect. The
amount that is "effective" will vary based on the organism to be
neutralized, whether an external surface is to be treated or whether the
composition is to be administered as a pharmaceutical, the mode of
administration, and the like. Thus, it is not always possible to specify
an exact "effective amount." However, an appropriate "effective" amount
in any individual case may be determined by one of ordinary skill in the
art using routine experimentation.

[0184] In one embodiment the method comprises contacting the bacteria with
an interferon-inducible (ELR-) CXC chemokine at a concentration of about
1 to about 100 μg/ml, about 1 to about 75 μg/ml, about 1 to about
50 μg/ml, 1 to about 30 μg/ml, 1 to about 15 μg/ml, 2 to about
10 μg/ml, 4 to about 8 μg/ml, 6 to about 10 μg/ml or about 8
μg/ml. Typically the bacteria are contacted with an effective amount
of the interferon-inducible (ELR-) CXC chemokines for a time ranging from
1 to 6, 2 to 8, 4 to 12 or 12 to 24 hours.

[0185] In accordance with one embodiment the administered anti-microbial
composition comprises an interferon-inducible (ELR-) CXC chemokine having
a peptide sequence selected from the group consisting of SEQ ID NO: 3,
SEQ ID NO: 6 and SEQ ID NO: 9, or a peptidomimetic derivative thereof. In
one embodiment such a composition is used to neutralize and/or kill both
active and stationary phase pathogenic bacteria, including for example
pathogenic organism is selected from the group consisting of
Streptococcus pneumoniae, Staphylococcus aureus, Moraxella catarrhalis,
Hemophilus influenzae, Enterobacteriaceae, Pseudomonas aeruginosa,
Stenotrophomonas maltophilia, Streptococcus viridans, Neisseria spp., and
Corynebacterium spp.

[0186] Several disease states are associated with large populations of
stationary phase bacteria, and currently there are not effective
treatments for removing such bacterial colonizations of patients. These
diseases include pneumonia (this is especially applicable to
ventilator-associated pneumonias but also applies to community-acquired
pneumonias), and pulmonary infections associated with, chronic
obstructive pulmonary disease (COPD), cystic fibrosis (CF), wherein
populations of bacteria remain resident in the host organism. Major
contributors to pathogenic infections of patient airways include both
Gram-positive and Gram-negative bacteria and include, but are not limited
to the following as major contributors Gram-positive cocci such as
Streptococcus and Staphylococcus species, including for example
Streptococcus pneumoniae and Staphylococcus aureus, Gram-negative cocci
such as Moraxella catarrhalis, Gram-negative rods such as Hemophilus
influenzae, Enterobacteriaceae, and Pseudomonas aeruginosa. Additional
organisms that might play a role in immunocompromised hosts (in addition
to the above listed organisms) may include Streptococcus viridans group,
coagulase-negative staphylococci, Neisseria spp., and Corynebacterium
spp. Yeast such as Candida spp. can also play a role. In cystic fibrosis
patients, Stenotrophomonas maltophilia is an ever more problematic Gram
negative pathogen that colonizes the airways along with the above listed
organisms (especially Pseudomonas aeruginosa and S. aureus). One aspect
of the present disclosure is the use of the interferon-inducible (ELR-)
CXC chemokines to treat subjects suffering from a disease or condition
that is exacerbated by the presence of inappropriate bacteria such as
those listed above.

[0189] In one embodiment the compositions further comprise additional
anti-microbial agents including, for example, one or more antibiotics. In
another embodiment the method comprises administering one or more
interferon-inducible (ELR-) CXC chemokine peptides wherein the
interferon-inducible (ELR-) CXC chemokine is linked, optionally via
covalent bonding and optionally via a linker, to a conjugate moiety.
Linkage can be accomplished by covalent chemical bonds, physical forces
such electrostatic, hydrogen, ionic, van der Waals, or hydrophobic or
hydrophilic interactions. A variety of non-covalent coupling systems may
be used, including biotin-avidin, ligand/receptor, enzyme/substrate,
nucleic acid/nucleic acid binding protein, lipid/lipid binding protein,
cellular adhesion molecule partners; or any binding partners or fragments
thereof which have affinity for each other.

[0190] The peptide can be linked to conjugate moieties via direct covalent
linkage by reacting targeted amino acid residues of the peptide with an
organic derivatizing agent that is capable of reacting with selected side
chains or the N- or C-terminal residues of these targeted amino acids.
Reactive groups on the peptide or conjugate include, e.g., an aldehyde,
amino, ester, thiol, α-haloacetyl, maleimido or hydrazino group.
Derivatizing agents include, for example, maleimidobenzoyl
sulfosuccinimide ester (conjugation through cysteine residues),
N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic
anhydride or other agents known in the art. Alternatively, the conjugate
moieties can be linked to the peptide indirectly through intermediate
carriers, such as polysaccharide or polypeptide carriers. Examples of
polysaccharide carriers include aminodextran. Examples of suitable
polypeptide carriers include polylysine, polyglutamic acid, polyaspartic
acid, co-polymers thereof, and mixed polymers of these amino acids and
others, e.g., serines, to confer desirable solubility properties on the
resultant loaded carrier.

[0191] Exemplary conjugate moieties that can be linked to any of the
glucagon peptides described herein include but are not limited to a
heterologous peptide or polypeptide (including for example, a plasma
protein), a targeting agent, an immunoglobulin or portion thereof (e.g.
variable region, CDR, or Fc region), a diagnostic label such as a
radioisotope, fluorophore or enzymatic label, a polymer including water
soluble polymers, or other therapeutic or diagnostic agents.

[0192] In accordance with one embodiment a method of treating a pathogenic
colonization of a patient is provided wherein a composition comprising
the interferon-inducible (ELR-) CXC chemokine linked to a lipid vesicle
is administered to a subject in need thereof. In one embodiment the
interferon-inducible (ELR-) CXC chemokine is linked to the external
surface of the lipid vesicle, and in one embodiment the
interferon-inducible (ELR-) CXC chemokine is covalently bound to the
lipids comprising the lipid vesicle. In an alternative embodiment the
interferon-inducible (ELR-) CXC chemokine is entrapped within the lipid
vesicle. In one embodiment the lipid vesicle is a liposome. In a further
embodiment the composition comprises additional anti-microbial agents,
including for example one or more antibiotics. It is anticipated that the
administration of the interferon-inducible (ELR-) CXC chemokine will
enhance the efficacy of the known anti-microbial agent. The known
anti-microbial agents can be co-administered with the
interferon-inducible (ELR-) CXC chemokine either in a single dosage form
or the therapeutic agents can be administered sequentially, within 5, 10,
15, 30, 60, 120, 180, 240 minutes or 12, 24 or 48 hours, to one another.
In one embodiment the interferon-inducible (ELR-) CXC chemokine is linked
to a liposome, optionally with the known anti-microbial agents also
linked to the same liposome.

Neutralizing Multi-Drug Resistant Strains

[0193] During the last several decades bacterial resistance has emerged as
a new trend, contributing to morbidity and mortality caused by bacterial
infections. A troubling percentage of bacterial pathogens causing
infections encountered in clinical settings are resistant to some form of
antibiotic therapy. Due to the excessive and not always appropriate use
of antibiotics in humans and animal feed, bacterial resistance currently
constitutes a major public health crisis. The World Health Organization
(WHO) reported that drug resistant strains of microbes had a negative
impact on their fight against tuberculosis, cholera, diarrhea and
pneumonia, which together killed more than ten million people worldwide
in 1995.

[0194] Multi-drug resistant strains of bacteria such as
methicillin-resistant Staphylococcal aureus (MRSA) and
vancomycin-resistant enterococci (VRE) were first encountered in hospital
settings, but many of them can now be found infecting healthy individuals
in larger communities. The spread of VRE is particularly concerning when
it is taken into account that vancomycin is generally regarded as the
last line of defense in the antibiotic arsenal. Additionally, the
extensive use of beta-lactam antibiotics such as penicillin and
ampicillin has also resulted in significant numbers of resistant strains
among both Gram-positive and Gram-negative bacteria. Furthermore, strains
can be deliberately engineered to have multi-drug resistance as part of
"weaponization" of wild type strains, including for example Bacillus
anthracis.

[0195] Currently, the choices for treatment of antibiotic-resistant and
multi-drug resistant bacteria are limited in scope even though the
molecular mechanisms of resistance are fairly well understood. The four
main mechanisms by which microorganisms exhibit resistance to
antimicrobials are:

[0196] 1) Drug inactivation or modification: e.g. enzymatic deactivation
of Penicillin G in some penicillin-resistant bacteria through the
production of β-lactamases.

[0197] 2) Alteration of target site: e.g. alteration of PBP--the binding
target site of penicillins--in MRSA and other penicillin-resistant
bacteria.

[0198] 3) Alteration of metabolic pathway: e.g. some sulfonamide-resistant
bacteria do not require para-aminobenzoic acid (PABA), an important
precursor for the synthesis of folic acid and nucleic acids in bacteria
inhibited by sulfonamides. Instead, like mammalian cells, they turn to
utilizing preformed folic acid.

[0199] 4) Reduced drug accumulation: by decreasing drug permeability
and/or increasing active efflux (pumping out) of the drugs across the
cell surface. In many cases, antibiotic-resistant and multi-drug
resistant bacteria such as MRSA and VRE encode the antibiotic resistance
genes on plasmids. These plasmids can be laterally transferred between
bacteria and hence account for the rapid dissemination of antibiotic
resistance genes into diverse bacterial populations.

[0200] Surprisingly, applicants have found that compositions comprising
the interferon-inducible (ELR-) CXC chemokines disclosed herein have
efficacy in neutralizing multi-drug resistant bacteria. Accordingly, one
aspect of the present disclosure is the use of the interferon-inducible
(ELR-) CXC chemokines either alone or in combination with other
anitmicrobial agents to neutralize multi-drug resistant bacteria. In one
embodiment a method for inhibiting the proliferation of a multi-drug
resistant bacteria comprises contacting a multi-drug resistant bacteria
with an effective amount of the compound of an interferon-inducible
(ELR-) CXC chemokine of the present disclosure.

[0202] In one embodiment the method of neutralizing multi-drug resistant
bacteria comprises contacting the bacteria with an interferon-inducible
(ELR-) CXC chemokine at a concentration of about 1 to about 100 μg/ml,
about 1 to about 75 μg/ml, about 1 to about 50 μg/ml, 1 to about 30
μg/ml, 1 to about 15 μg/ml, 2 to about 10 μg/ml, 4 to about 8
μg/ml, 6 to about 10 μg/ml or about 8 μg/ml.

[0203] Since interferons are known to induce expression of native CXCL9,
CXCL10 and CXCL11, in one embodiment the method of treatment comprises
the co-administration to a subject in need thereof one or more
interferons, including for example interferon-alpha, interferon-beta
and/or interferon-gamma as an adjuvant to promote production of native

[0204] CXCL9, CXCL10 and CXCL11 chemokines in vivo. Co-aministration can
be accomplished by simultaneously administering the chemokine and the
interferon, or the two active agents can be administered one after the
other within 1, 2, 3, 4, 5, 6, 12, 24 or 48 hours of each other.

Neutralizing Bacterial Spores

[0205] Spores are resistant to most agents that would normally kill the
vegetative cells they formed from. Household cleaning products generally
have no effect, nor do most alcohols, quaternary ammonium compounds or
detergents. Currently, treatments are not available that are designed to
decontaminate (e.g., neutralize and/or prevent the growth or germination
of) spores on human skin or other human surfaces (e.g., lungs or hair).
Thus, there is a need for compositions and methods that can neutralize
and prevent the outgrowth of spores of pathogenic bacteria such as
Bacillus anthracis. Such an agent would ideally be easily disseminated,
not be harmful to human surfaces (e.g., skin or lungs) and would be
capable of altering (e.g., inhibiting) spore germination and growth
potential (e.g., thereby leaving the spores inert and non-infectious).

[0206] Surprisingly, applicants have discovered that interferon-inducible
(ELR-) CXC chemokines are effective in neutralizing spores. Specifically,
recombinant CXCL9, CXCL10, and CXCL11 exhibit direct inhibitory effects
on spore germination and directly kill vegetative cells of B. anthracis
(See FIGS. 10A & 10B). Furthermore, selective in vivo neutralization of
CXCL9 or CXCL9/CXCL10, or CXCL9/CXCL10/CXCL11 rendered normally resistant
C57BL/6 mice susceptible to pulmonary anthrax, whereas neutralization of
their shared receptor, CXCR3 (i.e., the common receptor expressed on
leukocytes recruited to the site of infection by CXCL9, CXCL10, CXCL11),
had no impact on survival. These findings support the notion that
interferon-inducible (ELR-) CXC chemokines have direct antimicrobial
effects against B. anthracis in vitro and during in vivo infection.

[0207] In accordance with one embodiment a method of neutralizing spores,
particularly of pathogenic bacteria such as B. anthracis and C. difficile
is provided, wherein the method comprises contacting the spores with a
composition comprising an interferon-inducible (ELR-) CXC chemokine In
accordance with one embodiment the method comprises the steps of
contacting the spores with an effective amount of a peptide selected from
the group consisting of i) CXCL-9 (SEQ ID NO: 1), CXCL-10 (SEQ ID NO: 4)
or CXCL 11 (SEQ ID NO: 7), ii) a peptide fragment of CXCL-9, CXCL-10 or
CXCL 11, or a peptide having at least 90% amino acid sequence identity
with i) or ii). In one embodiment the peptide comprises the sequence of
SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 9, or a peptidomimetic
derivative thereof. It is anticipated that compositions comprising the
interferon-inducible (ELR-) CXC chemokines disclosed herein can be
formulated for treating external surfaces (e g skin or hair) or can be
formulated as pharmaceuticals for administration (e.g. inhaled
formulations) to subjects to neutralize internalized (e.g., the lungs)
spores in vivo.

[0209] The present invention is not limited by the type of bacterial spore
neutralized. In some embodiments, the spore is a Bacillus spore,
including for example a Bacillus anthracis spore. The Bacillus anthracis
spore may be a naturally occurring spore or a genetically or mechanically
engineered form. The spore may also be from an antibiotic resistant
strain of B. anthracis (e.g., ciprofloxacin resistant). In some
embodiments, the interferon-inducible (ELR-) CXC chemokine is
administered to a subject under conditions such that spore germination or
growth is prohibited and/or attenuated. In some embodiments, greater than
70%, 80%, or 90% of bacterial spores are neutralized (e.g., killed). In
some embodiments, there is greater than 2 log (e.g., greater than 3 log,
4 log, 5 log, . . .) reduction in bacterial spore outgrowth. In some
embodiments, reduction in spore outgrowth occurs within hours (e.g., with
1 hour (e.g., in 20-40 minutes or less), within 2 hours, within 3 hours,
within 6 hours or within 12 hours). In some embodiments, neutralization
of the spore (e.g., the inability of the spore to germinate) lasts for at
least 3 days, at least 7 days, at least 14 days, at least 21 days, at
least 28 days, or at least 56 days.

[0210] In one embodiment the method comprises contacting the spores with
an interferon-inducible (ELR-) CXC chemokine at a concentration of about
1 to about 100 μg/ml, about 1 to about 75 μg/ml, about 1 to about
50 μg/ml, 1 to about 30 μg/ml, 1 to about 15 μg/ml, 2 to about
10 μg/ml, 4 to about 8 μg/ml, 6 to about 10 μg/ml or about 8
μg/ml.

[0220] Of note, C. difficile is one of the most problematic spore-forming
pathogens in hospitalized patients since it can cause severe diarrhea and
even colonic rupture. Emergence of hypervirluent strains has occurred
over the past few years with an observed higher mortality.

[0221] Surprisingly, applicants have found that the interferon-inducible
(ELR-) CXC chemokines have activity in neutralizing spores under
physiological conditions. In accordance with one embodiment a method is
provided for neutralizing spores of a prokaryotic pathogenic organism.
The method comprises contacting the spores with a composition comprising
an interferon-inducible (ELR-) CXC chemokine. In accordance with one
embodiment a method is provided for neutralizing spores from an organism
selected from the group consisting of Bacillus anthracis, Bacillus
cereus, Clostridium difficile, Clostridium botulinum, Clostridium
perfringens, Clostridium tetani and Clostridium sordellii. In one
embodiment the method comprises neutralizing spores from an organism
selected from the group consisting of Bacillus anthracis and Clostridium
difficile, and in one specific embodiment the method comprises
neutralizing Bacillus anthracis spores.

[0223] Since interferons are known to induce expression of native CXCL9,
CXCL10 and CXCL11, in one embodiment the method of treating a patient who
has come in contact with spores comprises the co-administration of one or
more interferons, including for example interferon-alpha, interferon-beta
and/or interferon-gamma as an adjuvant to promote production of native
CXCL9, CXCL10 and CXCL11 chemokines in vivo. Co-aministration can be
accomplished by simultaneously administering the chemokine and the
interferon, or the two active agents can be administered one after the
other within 1, 2, 3, 4, 5, 6, 12, 24 or 48 hours of each other.

[0224] In one embodiment the compositions further comprise additional
anti-microbial agents including, for example, one or more antibiotics. In
another embodiment the method comprises administering one or more
interferon-inducible (ELR-) CXC chemokine peptides wherein the
interferon-inducible (ELR-) CXC chemokine is linked, optionally via
covalent bonding and optionally via a linker, to a conjugate moiety.
Linkage can be accomplished by covalent chemical bonds, physical forces
such electrostatic, hydrogen, ionic, van der Waals, or hydrophobic or
hydrophilic interactions. A variety of non-covalent coupling systems may
be used, including biotin-avidin, ligand/receptor, enzyme/substrate,
nucleic acid/nucleic acid binding protein, lipid/lipid binding protein,
cellular adhesion molecule partners; or any binding partners or fragments
thereof which have affinity for each other.

[0225] The peptide can be linked to conjugate moieties via direct covalent
linkage by reacting targeted amino acid residues of the peptide with an
organic derivatizing agent that is capable of reacting with selected side
chains or the N- or C-terminal residues of these targeted amino acids.
Reactive groups on the peptide or conjugate include, e.g., an aldehyde,
amino, ester, thiol, a-haloacetyl, maleimido or hydrazino group.
Derivatizing agents include, for example, maleimidobenzoyl
sulfosuccinimide ester (conjugation through cysteine residues),
N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic
anhydride or other agents known in the art. Alternatively, the conjugate
moieties can be linked to the peptide indirectly through intermediate
carriers, such as polysaccharide or polypeptide carriers. Examples of
polysaccharide carriers include aminodextran. Examples of suitable
polypeptide carriers include polylysine, polyglutamic acid, polyaspartic
acid, co-polymers thereof, and mixed polymers of these amino acids and
others, e.g., serines, to confer desirable solubility properties on the
resultant loaded carrier.

[0226] Exemplary conjugate moieties that can be linked to any of the
glucagon peptides described herein include but are not limited to a
heterologous peptide or polypeptide (including for example, a plasma
protein), a targeting agent, an immunoglobulin or portion thereof (e.g.
variable region, CDR, or Fc region), a diagnostic label such as a
radioisotope, fluorophore or enzymatic label, a polymer including water
soluble polymers, or other therapeutic or diagnostic agents.

[0227] In accordance with one embodiment a method of neutralizing spores
is provided wherein a composition comprising an interferon-inducible
(ELR-) CXC chemokine linked to a lipid vesicle is administered to a
subject in need thereof. In one embodiment the interferon-inducible
(ELR-) CXC chemokine is linked to the external surface of the lipid
vesicle, and in one embodiment the interferon-inducible (ELR-) CXC
chemokine is covalently bound to the lipids comprising the lipid vesicle.
In an alternative embodiment the interferon-inducible (ELR-) CXC
chemokine is entrapped within the lipid vesicle. In one embodiment the
lipid vesicle is a liposome. In a further embodiment the composition
comprises additional anti-microbial agents, including for example one or
more antibiotics. It is anticipated that the administration of the
interferon-inducible (ELR-) CXC chemokine will enhance the efficacy of
the known anti-microbial agent. The known anti-microbial agents can be
co-administered with the interferon-inducible (ELR-) CXC chemokine either
in a single dosage form or the therapeutic agents can be administered
sequentially, within 5, 10, 15, 30, 60, 120, 180, 440 minutes or 12, 24
or 48 hours, to one another. In one embodiment the interferon-inducible
(ELR-) CXC chemokine is linked to a liposome, optionally with the known
anti-microbial agents also linked to the same liposome.

[0228] In accordance with one embodiment the interferon-inducible (ELR-)
CXC chemokine compositions disclosed herein are used to treat solid
surfaces to neutralize spore contaminated surfaces. In one embodiment the
compositions disclosed herein are used to decontaminate organic materials
including food or the external surfaces of animals including human skin.
In another embodiment the methods for neutralizing spores comprises
administering a pharmaceutical composition comprising an
interferon-inducible (ELR-) CXC chemokine to neutralize spores that have
been internalized by a subject. In one embodiment the composition is
formulated as an aerosol, mist, fine powder or other formulation known to
those skilled in the art for administration to pulmonary system. In one
embodiment the composition is formulated for oral delivery using
formulations known to those skilled in the art for administration to the
digestive tract.

Methods of Identifying Antagonists and Inhibitors of FtsX

[0229] As used herein, an antagonist or inhibiting agent may comprise,
without limitation, a drug, a small molecule, an antibody, an antigen
binding portion thereof or a biosynthetic antibody binding site that
binds a particular target protein; an antisense molecule that hybridizes
in vivo to a nucleic acid encoding a target protein or a regulatory
element associated therewith, or a ribozyme, aptamer, a phylomer or small
molecule that binds to and/or inhibits a target protein, or that binds to
and/or inhibits, reduces or otherwise modulates expression of nucleic
acid encoding a target protein, including for example RNA interference
(e.g., use of small interfering RNA (siRNA)).

[0230] This invention encompasses methods of screening compounds to
identify those compounds that act as agonists (stimulate) or antagonists
(inhibit) of the protein interactions and pathways described herein.
Screening assays for antagonist compound candidates are designed to
identify compounds that bind or complex with the peptides described
herein, or otherwise interfere with the interaction of the peptides with
other proteins. Such screening assays will include assays amenable to
high-throughput screening of chemical libraries, making them particularly
suitable for identifying small molecule drug candidates.

[0231] FtsX assays also include those described in detail herein, such as
far-western, co-immunoprecipitation, immunoassays,
immunocytochemical/immuno localization, interaction with FtsX protein,
fertilization, contraception, and immunogenicity.

[0232] The assays can be performed in a variety of formats, including
protein-protein binding assays, biochemical screening assays,
high-throughput assays, immunoassays, and cell-based assays, which are
well characterized in the art.

[0233] All assays for antagonists are common in that they call for
contacting the compound or drug candidate with a peptide identified
herein under conditions and for a time sufficient to allow these two
components to interact.

[0234] In binding assays, the interaction is binding and the complex
formed can be isolated or detected in the reaction mixture. In a
particular embodiment, one of the peptides of the complexes described
herein, or the test compound or drug candidate is immobilized on a solid
phase, e.g., on a microtiter plate, by covalent or non-covalent
attachments. Non-covalent attachment generally is accomplished by coating
the solid surface with a solution of the peptide and drying.
Alternatively, an immobilized antibody, e.g., a monoclonal antibody,
specific for the peptide to be immobilized can be used to anchor it to a
solid surface. The assay is performed by adding the non-immobilized
component, which may be labeled by a detectable label, to the immobilized
component, e.g., the coated surface containing the anchored component.
When the reaction is complete, the non-reacted components are removed,
e.g., by washing, and complexes anchored on the solid surface are
detected. When the originally non-immobilized component carries a
detectable label, the detection of label immobilized on the surface
indicates that complexing occurred. Where the originally non-immobilized
component does not carry a label, complexing can be detected, for
example, by using a labeled antibody specifically binding the immobilized
complex.

[0235] If the candidate compound interacts with, but does not bind to a
particular peptide identified herein, its interaction with that peptide
can be assayed by methods well known for detecting protein-protein
interactions. Such assays include traditional approaches, such as, e.g.,
cross-linking, co-immunoprecipitation, and co-purification through
gradients or chromatographic columns. In addition, protein-protein
interactions can be monitored by using a yeast-based genetic system
described by Fields and co-workers (Fields and Song, Nature (London),
340:245-246 (1989); Chien et al., Proc. Natl. Acad. Sci. USA,
88:9578-9582 (1991)) as disclosed by Chevray and Nathans, Proc. Natl.
Acad. Sci. USA, 89: 5789-5793 (1991). Complete kits for identifying
protein-protein interactions between two specific proteins using the
two-hybrid technique are available. This system can also be extended to
map protein domains involved in specific protein interactions as well as
to pinpoint amino acid residues that are crucial for these interactions.

[0236] Compounds that interfere with the interaction of a peptide
identified herein and other intra- or extracellular components can be
tested as follows: usually a reaction mixture is prepared containing the
product of the gene and the intra- or extracellular component under
conditions and for a time allowing for the interaction and binding of the
two products. To test the ability of a candidate compound to inhibit
binding, the reaction is run in the absence and in the presence of the
test compound. In addition, a placebo may be added to a third reaction
mixture, to serve as positive control. The binding (complex formation)
between the test compound and the intra- or extracellular component
present in the mixture is monitored as described hereinabove. The
formation of a complex in the control reaction(s) but not in the reaction
mixture containing the test compound indicates that the test compound
interferes with the interaction of the test compound and its reaction
partner.

[0237] To assay for antagonists, the peptide may be added to a cell along
with the compound to be screened for a particular activity and the
ability of the compound to inhibit the activity of interest in the
presence of the peptide indicates that the compound is an antagonist to
the peptide. The peptide can be labeled, such as by radioactivity.

[0238] Other assays and libraries are encompassed within the invention,
such as the use of phylomers® and reverse yeast two-hybrid assays
(see Watt, 2006, Nature Biotechnology, 24:177; Watt, U.S. Pat. No.
6,994,982; Watt, U.S. Pat. Pub. No. 2005/0287580; Watt, U.S. Pat. No.
6,510,495; Barr et al., 2004, J. Biol. Chem., 279:41:43178-43189; the
contents of each of these publications is hereby incorporated by
reference herein in their entirety). Phylomers® are derived from sub
domains of natural proteins, which makes them potentially more stable
than conventional short random peptides. Phylomers® are sourced from
biological genomes that are not human in origin. This feature
significantly enhances the potency associated with Phylomers® against
human protein targets. Phylogica's current Phylomer® library has a
complexity of 50 million clones, which is comparable with the numerical
complexity of random peptide or antibody Fab fragment libraries. An
Interacting Peptide Library, consisting of 63 million peptides fused to
the B42 activation domain, can be used to isolate peptides capable of
binding to a target protein in a forward yeast two hybrid screen. The
second is a Blocking Peptide Library made up of over 2 million peptides
that can be used to screen for peptides capable of disrupting a specific
protein interaction using the reverse two-hybrid system.

[0239] The Phylomer® library consists of protein fragments, which have
been sourced from a diverse range of bacterial genomes. The libraries are
highly enriched for stable subdomains (15-50 amino acids long). This
technology can be integrated with high throughput screening techniques
such as phage display and reverse yeast two-hybrid traps.

[0240] The present application discloses compositions and methods for
inhibiting the proteins described herein, and those not disclosed which
are known in the art are encompassed within the invention. For example,
various modulators/effectors are known, e.g. antibodies, biologically
active nucleic acids, such as antisense molecules, RNAi molecules, or
ribozymes, aptamers, peptides or low-molecular weight organic compounds
recognizing said polynucleotides or polypeptides.

[0241] The present application also encompasses pharmaceutical and
therapeutic compositions comprising the compounds of the present
invention.

[0242] The present application is also directed to pharmaceutical
compositions comprising the peptides of the present invention. More
particularly, such compounds can be formulated as pharmaceutical
compositions using standard pharmaceutically acceptable carriers,
fillers, solublizing agents and stabilizers known to those skilled in the
art. The pharmaceutical compositions can be formulated to be administered
using standard routes of administration including for example, oral,
parenteral, topical and as an inhaled formulation, using standard
formulations and techniques known to those skilled in the art.

[0245] Also encompassed by the present disclosures are antibodies raised
against the proteins and peptides disclosed herein. The generation of
polyclonal antibodies is accomplished by inoculating the desired animal
with the antigen and isolating antibodies which specifically bind the
antigen therefrom.

[0247] The present disclosure also encompasses the use pharmaceutical
compositions of an appropriate compound, homo log, fragment, analog, or
derivative thereof to practice the methods of the invention, the
composition comprising at least one appropriate compound, homo log,
fragment, analog, or derivative thereof and a pharmaceutically-acceptable
carrier.

[0248] The pharmaceutical compositions useful for practicing the invention
may be administered to deliver a dose of between 1 ng/kg/day and 100
mg/kg/day. Pharmaceutical compositions that are useful in the methods of
the invention may be administered systemically in oral solid
formulations, ophthalmic, suppository, aerosol, topical or other similar
formulations. In addition to the appropriate compound, such
pharmaceutical compositions may contain pharmaceutically-acceptable
carriers and other ingredients known to enhance and facilitate drug
administration. Other possible formulations, such as nanoparticles,
liposomes, resealed erythrocytes, and immunologically based systems may
also be used to administer an appropriate compound according to the
methods of the invention.

[0249] Compounds which are identified using any of the methods described
herein may be formulated and administered to a mammal for treatment of
the diseases disclosed herein are now described.

[0250] The invention encompasses the preparation and use of pharmaceutical
compositions comprising a compound useful for treatment of the
conditions, disorders, and diseases disclosed herein as an active
ingredient. Such a pharmaceutical composition may consist of the active
ingredient alone, in a form suitable for administration to a subject, or
the pharmaceutical composition may comprise the active ingredient and one
or more pharmaceutically acceptable carriers, one or more additional
ingredients, or some combination of these. The active ingredient may be
present in the pharmaceutical composition in the form of a
physiologically acceptable ester or salt, such as in combination with a
physiologically acceptable cation or anion, as is well known in the art.

[0251] As used herein, the term "physiologically acceptable" ester or salt
means an ester or salt form of the active ingredient which is compatible
with any other ingredients of the pharmaceutical composition, which is
not deleterious to the subject to which the composition is to be
administered.

[0252] The formulations of the pharmaceutical compositions described
herein may be prepared by any method known or hereafter developed in the
art of pharmacology. In general, such preparatory methods include the
step of bringing the active ingredient into association with a carrier or
one or more other accessory ingredients, and then, if necessary or
desirable, shaping or packaging the product into a desired single- or
multi-dose unit.

[0253] Although the descriptions of pharmaceutical compositions provided
herein are principally directed to pharmaceutical compositions which are
suitable for ethical administration to humans, it will be understood by
the skilled artisan that such compositions are generally suitable for
administration to animals of all sorts. Modification of pharmaceutical
compositions suitable for administration to humans in order to render the
compositions suitable for administration to various animals is well
understood, and the ordinarily skilled veterinary pharmacologist can
design and perform such modification with merely ordinary, if any,
experimentation. Subjects to which administration of the pharmaceutical
compositions of the invention is contemplated include, but are not
limited to, humans and other primates, mammals including commercially
relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs,
birds including commercially relevant birds such as chickens, ducks,
geese, and turkeys.

[0254] Pharmaceutical compositions that are useful in the methods of the
invention may be prepared, packaged, or sold in formulations suitable for
oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal,
buccal, ophthalmic, intrathecal or another route of administration. Other
contemplated formulations include projected nanoparticles, liposomal
preparations, resealed erythrocytes containing the active ingredient, and
immunologically-based formulations. The pharmaceutical compositions of
the present invention can be processed into a tablet form, capsule form,
or suspension that is suited for oral administration or can be
reconstituted in an aqueous solvent (e.g., DI water or saline) for oral,
IV, or inhalation (e.g., nebulizer) administration.

[0255] A pharmaceutical composition of the invention may be prepared,
packaged, or sold in bulk, as a single unit dose, or as a plurality of
single unit doses. As used herein, a "unit dose" is discrete amount of
the pharmaceutical composition comprising a predetermined amount of the
active ingredient. The amount of the active ingredient is generally equal
to the dosage of the active ingredient which would be administered to a
subject or a convenient fraction of such a dosage such as, for example,
one-half or one-third of such a dosage.

[0256] The relative amounts of the active ingredient, the pharmaceutically
acceptable carrier, and any additional ingredients in a pharmaceutical
composition of the invention will vary, depending upon the identity,
size, and condition of the subject treated and further depending upon the
route by which the composition is to be administered. By way of example,
the composition may comprise between 0.1% and 100% (w/w) active
ingredient.

[0257] In addition to the active ingredient, a pharmaceutical composition
of the invention may further comprise one or more additional
pharmaceutically active agents. Particularly contemplated additional
agents include anti-emetics and scavengers such as cyanide and cyanate
scavengers.

[0258] Controlled- or sustained-release formulations of a pharmaceutical
composition of the invention may be made using conventional technology. A
formulation of a pharmaceutical composition of the invention suitable for
oral administration may be prepared, packaged, or sold in the form of a
discrete solid dose unit including, but not limited to, a tablet, a hard
or soft capsule, a cachet, a troche, or a lozenge, each containing a
predetermined amount of the active ingredient. Other formulations
suitable for oral administration include, but are not limited to, a
powdered or granular formulation, an aqueous or oily suspension, an
aqueous or oily solution, or an emulsion.

[0259] As used herein, an "oily" liquid is one which comprises a
carbon-containing liquid molecule and which exhibits a less polar
character than water.

[0260] A tablet comprising the active ingredient may, for example, be made
by compressing or molding the active ingredient, optionally with one or
more additional ingredients. Compressed tablets may be prepared by
compressing, in a suitable device, the active ingredient in a free
flowing form such as a powder or granular preparation, optionally mixed
with one or more of a binder, a lubricant, an excipient, a surface active
agent, and a dispersing agent. Molded tablets may be made by molding, in
a suitable device, a mixture of the active ingredient, a pharmaceutically
acceptable carrier, and at least sufficient liquid to moisten the
mixture. Pharmaceutically acceptable excipients used in the manufacture
of tablets include, but are not limited to, inert diluents, granulating
and disintegrating agents, binding agents, and lubricating agents. Known
dispersing agents include, but are not limited to, potato starch and
sodium starch glycollate. Known surface active agents include, but are
not limited to, sodium lauryl sulphate. Known diluents include, but are
not limited to, calcium carbonate, sodium carbonate, lactose,
microcrystalline cellulose, calcium phosphate, calcium hydrogen
phosphate, and sodium phosphate. Known granulating and disintegrating
agents include, but are not limited to, corn starch and alginic acid.
Known binding agents include, but are not limited to, gelatin, acacia,
pre-gelatinized maize starch, polyvinylpyrrolidone, and hydroxypropyl
methylcellulo se. Known lubricating agents include, but are not limited
to, magnesium stearate, stearic acid, silica, and talc.

[0261] Tablets may be non-coated or they may be coated using known methods
to achieve delayed disintegration in the gastrointestinal tract of a
subject, thereby providing sustained release and absorption of the active
ingredient. By way of example, a material such as glyceryl monostearate
or glyceryl distearate may be used to coat tablets. Further by way of
example, tablets may be coated using methods described in U.S. Pat. Nos.
4,256,108; 4,160,452; and 4,265,874 to form osmotically-controlled
release tablets. Tablets may further comprise a sweetening agent, a
flavoring agent, a coloring agent, a preservative, or some combination of
these in order to provide pharmaceutically elegant and palatable
preparation.

[0262] Hard capsules comprising the active ingredient may be made using a
physiologically degradable composition, such as gelatin. Such hard
capsules comprise the active ingredient, and may further comprise
additional ingredients including, for example, an inert solid diluent
such as calcium carbonate, calcium phosphate, or kaolin.

[0263] Soft gelatin capsules comprising the active ingredient may be made
using a physiologically degradable composition, such as gelatin. Such
soft capsules comprise the active ingredient, which may be mixed with
water or an oil medium such as peanut oil, liquid paraffin, or olive oil.

[0264] Liquid formulations of a pharmaceutical composition of the
invention which are suitable for oral administration may be prepared,
packaged, and sold either in liquid form or in the form of a dry product
intended for reconstitution with water or another suitable vehicle prior
to use.

[0266] Known dispersing or wetting agents include, but are not limited to,
naturally occurring phosphatides such as lecithin, condensation products
of an alkylene oxide with a fatty acid, with a long chain aliphatic
alcohol, with a partial ester derived from a fatty acid and a hexitol, or
with a partial ester derived from a fatty acid and a hexitol anhydride
(e.g. polyoxyethylene stearate, heptadecaethyleneoxycetanol,
polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan
monooleate, respectively). Known emulsifying agents include, but are not
limited to, lecithin and acacia. Known preservatives include, but are not
limited to, methyl, ethyl, or n-propyl para hydroxybenzoates, ascorbic
acid, and sorbic acid. Known sweetening agents include, for example,
glycerol, propylene glycol, sorbitol, sucrose, and saccharin. Known
thickening agents for oily suspensions include, for example, beeswax,
hard paraffin, and cetyl alcohol.

[0267] Liquid solutions of the active ingredient in aqueous or oily
solvents may be prepared in substantially the same manner as liquid
suspensions, the primary difference being that the active ingredient is
dissolved, rather than suspended in the solvent. Liquid solutions of the
pharmaceutical composition of the invention may comprise each of the
components described with regard to liquid suspensions, it being
understood that suspending agents will not necessarily aid dissolution of
the active ingredient in the solvent. Aqueous solvents include, for
example, water and isotonic saline. Oily solvents include, for example,
almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis,
olive, sesame, or coconut oil, fractionated vegetable oils, and mineral
oils such as liquid paraffin.

[0268] Powdered and granular formulations of a pharmaceutical preparation
of the invention may be prepared using known methods. Such formulations
may be administered directly to a subject, used, for example, to form
tablets, to fill capsules, or to prepare an aqueous or oily suspension or
solution by addition of an aqueous or oily vehicle thereto. Each of these
formulations may further comprise one or more of dispersing or wetting
agent, a suspending agent, and a preservative. Additional excipients,
such as fillers and sweetening, flavoring, or coloring agents, may also
be included in these formulations.

[0269] A pharmaceutical composition of the invention may also be prepared,
packaged, or sold in the form of oil in water emulsion or a water-in-oil
emulsion. The oily phase may be a vegetable oil such as olive or arachis
oil, a mineral oil such as liquid paraffin, or a combination of these.
Such compositions may further comprise one or more emulsifying agents
such as naturally occurring gums such as gum acacia or gum tragacanth,
naturally occurring phosphatides such as soybean or lecithin phosphatide,
esters or partial esters derived from combinations of fatty acids and
hexitol anhydrides such as sorbitan monooleate, and condensation products
of such partial esters with ethylene oxide such as polyoxyethylene
sorbitan monooleate. These emulsions may also contain additional
ingredients including, for example, sweetening or flavoring agents.

[0270] A pharmaceutical composition of the invention may also be prepared,
packaged, or sold in a formulation suitable for rectal administration,
vaginal administration, parenteral administration

[0271] The pharmaceutical compositions may be prepared, packaged, or sold
in the form of a sterile injectable aqueous or oily suspension or
solution. This suspension or solution may be formulated according to the
known art, and may comprise, in addition to the active ingredient,
additional ingredients such as the dispersing agents, wetting agents, or
suspending agents described herein. Such sterile injectable formulations
may be prepared using a non toxic parenterally acceptable diluent or
solvent, such as water or 1,3 butane diol, for example. Other acceptable
diluents and solvents include, but are not limited to, Ringer's solution,
isotonic sodium chloride solution, and fixed oils such as synthetic mono
or di-glycerides. Other parenterally-administrable formulations which are
useful include those which comprise the active ingredient in
microcrystalline form, in a liposomal preparation, or as a component of a
biodegradable polymer systems. Compositions for sustained release or
implantation may comprise pharmaceutically acceptable polymeric or
hydrophobic materials such as an emulsion, an ion exchange resin, a
sparingly soluble polymer, or a sparingly soluble salt.

[0272] Formulations suitable for topical administration include, but are
not limited to, liquid or semi liquid preparations such as liniments,
lotions, oil in water or water in oil emulsions such as creams, ointments
or pastes, and solutions or suspensions. Topically-administrable
formulations may, for example, comprise from about 1% to about 10% (w/w)
active ingredient, although the concentration of the active ingredient
may be as high as the solubility limit of the active ingredient in the
solvent. Formulations for topical administration may further comprise one
or more of the additional ingredients described herein.

[0273] A pharmaceutical composition of the invention may be prepared,
packaged, or sold in a formulation suitable for pulmonary administration
via the buccal cavity. Such a formulation may comprise dry particles
which comprise the active ingredient and which have a diameter in the
range from about 0.5 to about 7 nanometers, and preferably from about 1
to about 6 nanometers. Such compositions are conveniently in the form of
dry powders for administration using a device comprising a dry powder
reservoir to which a stream of propellant may be directed to disperse the
powder or using a self propelling solvent/powder dispensing container
such as a device comprising the active ingredient dissolved or suspended
in a low-boiling propellant in a sealed container. Preferably, such
powders comprise particles wherein at least 98% of the particles by
weight have a diameter greater than 0.5 nanometers and at least 95% of
the particles by number have a diameter less than 7 nanometers. More
preferably, at least 95% of the particles by weight have a diameter
greater than 1 nanometer and at least 90% of the particles by number have
a diameter less than 6 nanometers. Dry powder compositions preferably
include a solid fine powder diluent such as sugar and are conveniently
provided in a unit dose form.

[0274] Low boiling propellants generally include liquid propellants having
a boiling point of below 65° F. at atmospheric pressure.
Generally, the propellant may constitute 50 to 99.9% (w/w) of the
composition, and the active ingredient may constitute 0.1 to 20% (w/w) of
the composition. The propellant may further comprise additional
ingredients such as a liquid non-ionic or solid anionic surfactant or a
solid diluent (preferably having a particle size of the same order as
particles comprising the active ingredient).

[0275] Pharmaceutical compositions of the invention formulated for
pulmonary delivery may also provide the active ingredient in the form of
droplets of a solution or suspension (e.g., use of a nebulizer). Such
formulations may be prepared, packaged, or sold as aqueous or dilute
alcoholic solutions or suspensions, optionally sterile, comprising the
active ingredient, and may conveniently be administered using any
nebulization or atomization device. Such formulations may further
comprise one or more additional ingredients including, but not limited
to, a flavoring agent such as saccharin sodium, a volatile oil, a
buffering agent, a surface active agent, or a preservative such as
methylhydroxybenzoate. The droplets provided by this route of
administration preferably have an average diameter in the range from
about 0.1 to about 200 nanometers.

[0276] The formulations described herein as being useful for pulmonary
delivery are also useful for intranasal delivery of a pharmaceutical
composition of the invention. Another formulation suitable for intranasal
administration is a coarse powder comprising the active ingredient and
having an average particle from about 0.2 to 500 micrometers. Such a
formulation is administered in the manner in which snuff is taken i.e. by
rapid inhalation through the nasal passage from a container of the powder
held close to the nares.

[0277] Formulations suitable for nasal administration may, for example,
comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) of
the active ingredient, and may further comprise one or more of the
additional ingredients described herein.

[0278] A pharmaceutical composition of the invention may be prepared,
packaged, or sold in a formulation suitable for buccal administration.
Such formulations may, for example, be in the form of tablets or lozenges
made using conventional methods, and may, for example, 0.1 to 20% (w/w)
active ingredient, the balance comprising an orally dissolvable or
degradable composition and, optionally, one or more of the additional
ingredients described herein. Alternately, formulations suitable for
buccal administration may comprise a powder or an aerosolized or atomized
solution or suspension comprising the active ingredient. Such powdered,
aerosolized, or aerosolized formulations, when dispersed, preferably have
an average particle or droplet size in the range from about 0.1 to about
200 nanometers, and may further comprise one or more of the additional
ingredients described herein.

[0279] A pharmaceutical composition of the invention may be prepared,
packaged, or sold in a formulation suitable for ophthalmic
administration. Such formulations may, for example, be in the form of eye
drops including, for example, a 0.1/1.0% (w/w) solution or suspension of
the active ingredient in an aqueous or oily liquid carrier. Such drops
may further comprise buffering agents, salts, or one or more other of the
additional ingredients described herein. Other
ophthalmically-administrable formulations which are useful include those
which comprise the active ingredient in microcrystalline form or in a
liposomal preparation.

[0281] Typically, dosages of the compound of the invention which may be
administered to an animal, preferably a human, range in amount from 1
μg to about 100 g per kilogram of body weight of the subject. While
the precise dosage administered will vary depending upon any number of
factors, including but not limited to, the type of animal and type of
disease state being treated, the age of the animal and the route of
administration. Preferably, the dosage of the compound will vary from
about 1 mg to about 10 g per kilogram of body weight of the animal. More
preferably, the dosage will vary from about 10 mg to about 1 g per
kilogram of body weight of the subject.

[0282] The compound may be administered to a subject as frequently as
several times daily, or it may be administered less frequently, such as
once a day, once a week, once every two weeks, once a month, or even less
frequently, such as once every several months or even once a year or
less. The frequency of the dose will be readily apparent to the skilled
artisan and will depend upon any number of factors, such as, but not
limited to, the type and severity of the disease being treated, the type,
and age of the subject, etc.

[0283] The invention also includes a kit comprising a compound of the
invention and an instructional material which describes administering the
composition to a cell or a tissue of a subject. In another embodiment,
this kit comprises a (preferably sterile) solvent suitable for dissolving
or suspending the composition of the invention prior to administering the
compound to the subject. The invention also provides a kit for
identifying a regulator of the target molecule of the invention.

[0284] Without further description, it is believed that one of ordinary
skill in the art can, using the preceding description and the following
illustrative examples, make and utilize the compounds of the present
invention and practice the claimed methods. The following working
examples therefore, specifically point out the preferred embodiments of
the present invention, and are not to be construed as limiting in any way
the remainder of the disclosure.

[0285] In one embodiment, the present method of immunization comprises the
administration of a source of immunogenically active polypeptide
fragments, said polypeptide fragments being selected from FtsX protein
fragments and/or homologs thereof as defined herein before, said
polypeptide fragments comprising dominant CTL and/or HTL epitopes and
which fragments are between 18 and 45 amino acids in length. Peptides
having a length between 18 and 45 amino acids have been observed to
provide superior immunogenic properties as is described in WO 02/070006.
In accordance with one embodiment an antigenic composition is provided
comprising an isolated peptide having the sequence of SEQ ID NO: 10 or a
contiguous 8 amino acid fragment of SEQ ID NO: 10. In accordance with one
embodiment the antigenic composition further comprises an adjuvant.

[0286] Peptides may advantageously be chemically synthesized and may
optionally be (partially) overlapping and/or may also be ligated to other
molecules, peptides, or proteins. Peptides may also be fused to form
synthetic proteins, as in Welters et al. (Vaccine. 2004 Dec. 2;
23(3):305-11). It may also be advantageous to add to the amino- or
carboxy-terminus of the peptide chemical moieties or additional (modified
or D-) amino acids in order to increase the stability and/or decrease the
biodegradability of the peptide. To improve immunogenicity,
immuno-stimulating moieties may be attached, e.g. by lipidation or
glycosylation. To enhance the solubility of the peptide, addition of
charged or polar amino acids may be used, in order to enhance solubility
and increase stability in vivo.

[0287] For immunization purposes, the aforementioned immunogenic
polypeptides of the invention may also be fused with proteins, such as,
but not limited to, tetanus toxin/toxoid, diphtheria toxin/toxoid or
other carrier molecules. The polypeptides according to the invention may
also be advantageously fused to heatshock proteins, such as recombinant
endogenous (murine) gp96 (GRP94) as a carrier for immunodominant peptides
as described in (references: Rapp U K and Kaufmann S H, Int Immunol. 2004
April; 16(4):597-605; Zugel U, Infect Immun. 2001 June; 69(6):4164-7) or
fusion proteins with Hsp70 (Triebel et al; WO9954464).

[0288] The individual amino acid residues of the present immunogenic
(poly)peptides of the invention can be incorporated in the peptide by a
peptide bond or peptide bond mimetic. A peptide bond mimetic of the
invention includes peptide backbone modifications well known to those
skilled in the art. Such modifications include modifications of the amide
nitrogen, the alpha carbon, amide carbonyl, complete replacement of the
amide bond, extensions, deletions, or backbone cross-links. See,
generally, Spatola, Chemistry and Biochemistry of Amino Acids, Peptides
and Proteins, Vol. VII (Weinstein ed., 1983). Several peptide backbone
modifications are known and can be used in the practice of the invention.

[0289] Amino acid mimetics may also be incorporated in the polypeptides.
An "amino acid mimetic" as used here is a moiety other than a naturally
occurring amino acid that conformationally and functionally serves as a
substitute for an amino acid in a polypeptide of the present invention.
Such a moiety serves as a substitute for an amino acid residue if it does
not interfere with the ability of the peptide to elicit an immune
response against the native FtsX T cell epitopes. Amino acid mimetics may
include non-protein amino acids. A number of suitable amino acid mimetics
are known to the skilled artisan, they include cyclohexylalanine,
3-cyclohexylpropionic acid, L-adamantyl alanine, adamantylacetic acid and
the like. Peptide mimetics suitable for peptides of the present invention
are discussed by Morgan and Gainor, (1989) Ann. Repts. Med. Chem.
24:243-252.

[0290] In one embodiment, the present method comprises the administration
of a composition comprising one or more of the present immunogenic
polypeptides as defined herein above, and at least one excipient.
Excipients are well known in the art of pharmacy and may for instance be
found in textbooks such as Remington's pharmaceutical sciences, Mack
Publishing, 1995.

[0291] The present method for immunization may further comprise the
administration, and in one aspect, the co-administration, of at least one
adjuvant. Adjuvants may comprise any adjuvant known in the art of
vaccination and may be selected using textbooks like Current Protocols in
Immunology, Wiley Interscience, 2004.

[0292] Adjuvants are herein intended to include any substance or compound
that, when used, in combination with an antigen, to immunize a human or
an animal, stimulates the immune system, thereby provoking, enhancing or
facilitating the immune response against the antigen, preferably without
generating a specific immune response to the adjuvant itself. In one
aspect, adjuvants can enhance the immune response against a given antigen
by at least a factor of 1.5, 2, 2.5, 5, 10, or 20, as compared to the
immune response generated against the antigen under the same conditions
but in the absence of the adjuvant. Tests for determining the statistical
average enhancement of the immune response against a given antigen as
produced by an adjuvant in a group of animals or humans over a
corresponding control group are available in the art. The adjuvant
preferably is capable of enhancing the immune response against at least
two different antigens. The adjuvant of the invention will usually be a
compound that is foreign to a human, thereby excluding immunostimulatory
compounds that are endogenous to humans, such as e.g. interleukins,
interferons, and other hormones.

[0294] The methods of immunization of the present application further
encompass the administration, including the co-administration, of a CD40
binding molecule in order to enhance a CTL response and thereby enhance
the therapeutic effects of the methods and compositions of the invention.
The use of CD40 binding molecules is described in WO 99/61065,
incorporated herein by reference. The CD40 binding molecule is preferably
an antibody or fragment thereof or a CD40 Ligand or a variant thereof,
and may be added separately or may be comprised within a composition
according to the current invention. Such effective dosages will depend on
a variety of factors including the condition and general state of health
of the patient. Thus, dosage regimens can be determined and adjusted by
trained medical personnel to provide the optimum therapeutic or
prophylactic effect.

[0295] In the present method, the one or more immunogenic polypeptides are
typically administered at a dosage of about 1 ug/kg patient body weight
or more at least once. Often dosages are greater than 10 ug/kg. According
to the present invention, the dosages preferably range from 1 ug/kg to 1
mg/kg.

[0296] In one embodiment typical dosage regimens comprise administering a
dosage of 1-1000 ug/kg, more preferably 10-500 ug/kg, still more
preferably 10-150 ug/kg, once, twice or three times a week for a period
of one, two, three, four or five weeks. According to one embodiment,
10-100 ug/kg is administered once a week for a period of one or two
weeks.

[0297] The present method, in one aspect, comprises administration of the
present immunogenic polypeptides and compositions comprising them via the
injection, transdermal, or oral route. In another, embodiment of the
invention, the present method comprises vaginal administration of the
present immunogenic polypeptides and compositions comprising them.

[0298] Another aspect of this disclosure relates to a pharmaceutical
preparation comprising as the active ingredient the present source of a
polypeptide as defined herein before. More particularly pharmaceutical
preparation comprises as the active ingredient one or more of the
aforementioned immunogenic polypeptides selected from the group of FtsX
proteins, homologues thereof and fragments of said FtsX proteins and
homologs thereof, or, alternatively, a gene therapy vector as defined
herein above.

[0299] The present invention further provides a pharmaceutical preparation
comprising one or more of the immunogenic polypeptides of the invention.
The concentration of said polypeptide in the pharmaceutical composition
can vary widely, i.e., from less than about 0.1% by weight, usually being
at least about 1% by weight to as much as 20% by weight or more.

[0300] The composition may comprise a pharmaceutically acceptable carrier
in addition to the active ingredient. The pharmaceutical carrier can be
any compatible, non-toxic substance suitable to deliver the immunogenic
polypeptides or gene therapy vectors to the patient. For polypeptides,
sterile water, alcohol, fats, waxes, and inert solids may be used as the
carrier. Pharmaceutically acceptable adjuvants, buffering agents,
dispersing agents, and the like, may also be incorporated into the
pharmaceutical compositions.

[0301] In one embodiment, the present pharmaceutical composition comprises
an adjuvant, as defined in more detail herein before. Adjuvants useful
for incorporation in the present composition are preferably selected from
the group of ligands that are recognized by a Toll-like-receptor (TLR)
present on antigen presenting cells, including lipopeptides,
lipopolysaccharides, peptidoglycans, liopteichoic acids,
lipoarabinomannans, lipoproteins (from mycoplasma or spirochetes),
double-stranded RNA (poly I:C), unmethylated DNA, flagellin,
CpG-containing DNA, and imidazoquinolines, as well derivatives of these
ligands having chemical modifications. The routineer will be able to
determine the exact amounts of anyone of these adjuvants to be
incorporated in the present pharmaceutical preparations in order to
render them sufficiently immunogenic. According to another preferred
embodiment, the present pharmaceutical preparation may comprise one or
more additional ingredients that are used to enhance CTL immunity as
explained herein before. According to a particularly preferred
embodiment, the present pharmaceutical preparation comprises a CD40
binding molecule.

[0302] Methods of producing pharmaceutical compositions comprising
polypeptides are described in U.S. Pat. Nos. 5,789,543 and 6,207,718. The
preferred form depends on the intended mode of administration and
therapeutic application.

[0303] In one embodiment, the present immunogenic proteins or polypeptides
are administered by injection. The parenteral route for administration of
the polypeptide is in accordance with known methods, e.g. injection or
infusion by intravenous, intraperitoneal, intramuscular, intra-arterial,
subcutaneous, or intralesional routes. The protein or polypeptide may be
administered continuously by infusion or by bolus injection. A typical
composition for intravenous infusion could be made up to contain 10 to 50
ml of sterile 0.9% NaCl or 5% glucose optionally supplemented with a 20%
albumin solution and between 10 ug and 50 mg, preferably between 50 ug
and 10 mg, of the polypeptide. A typical pharmaceutical composition for
intramuscular injection would be made up to contain, for example, 1-10 ml
of sterile buffered water and between 10 ug and 50 mg, preferably between
50 ug and 10 mg, of the polypeptide of the present invention. Methods for
preparing parenterally administrable compositions are well known in the
art and described in more detail in various sources, including, for
example, Remington's Pharmaceutical Science (15th ed., Mack Publishing,
Easton, Pa., 1980) (incorporated by reference in its entirety for all
purposes).

[0304] For convenience, immune responses are often described in the
present invention as being either "primary" or "secondary" immune
responses. A primary immune response, which is also described as a
"protective" immune response, refers to an immune response produced in an
individual as a result of some initial exposure (e.g., the initial
"immunization") to a particular antigen. Such an immunization can occur,
for example, as the result of some natural exposure to the antigen (for
example, from initial infection by some pathogen that exhibits or
presents the antigen). Alternatively, the immunization can occur because
of vaccinating the individual with a vaccine containing the antigen. For
example, the vaccine can be a vaccine comprising one or more antigenic
epitopes or fragments of FtsX.

[0305] The vaccine can also be modified to express other immune activators
such as IL2, and co-stimulatory molecules, among others.

[0306] Another type of vaccine that can be combined with antibodies to an
antigen is a vaccine prepared from a cell lysate of interest, in
conjunction with an immunological adjuvant, or a mixture of lysates from
cells of interest plus DETOX® immunological adjuvant. Vaccine
treatment can be boosted with anti-antigen antibodies, with or without
additional chemotherapeutic treatment.

[0307] When used in vivo for therapy, the antibodies of the subject
invention are administered to the subject in therapeutically effective
amounts (i.e., amounts that have desired therapeutic effect). They will
normally be administered parenterally. The dose and dosage regimen will
depend upon the degree of the infection, the characteristics of the
particular antibody or immunotoxin used, e.g., its therapeutic index, the
patient, and the patient's history. Advantageously the antibody or
immunotoxin is administered continuously over a period of 1-2 weeks or
longer as indicated or needed. Optionally, the administration is made
during the course of adjunct therapy such as antimicrobial treatment, or
administration of tumor necrosis factor, interferon, or other
cytoprotective or immunomodulatory agent.

[0308] For parenteral administration, the antibodies will be formulated in
a unit dosage injectable form (solution, suspension, emulsion) in
association with a pharmaceutically acceptable parenteral vehicle. Such
vehicles are inherently nontoxic, and non-therapeutic. Examples of such
vehicle are water, saline, Ringer's solution, dextrose solution, and 5%
human serum albumin. Nonaqueous vehicles such as fixed oils and ethyl
oleate can also be used. Liposomes can be used as carriers. The vehicle
can contain minor amounts of additives such as substances that enhance
isotonicity and chemical stability, e.g., buffers and preservatives. The
antibodies will typically be formulated in such vehicles at
concentrations of about 1.0 mg/ml to about 10 mg/ml.

[0309] Use of IgM antibodies can be preferred for certain applications;
however, IgG molecules by being smaller can be more able than IgM
molecules to localize to certain types of infected cells.

[0310] There is evidence that complement activation in vivo leads to a
variety of biological effects, including the induction of an inflammatory
response and the activation of macrophages (Unanue and Benecerraf,
Textbook of Immunology, 2nd Edition, Williams & Wilkins, p. 218 (1984)).
The increased vasodilation accompanying inflammation can increase the
ability of various agents to localize. Therefore, antigen-antibody
combinations of the type specified by this invention can be used in many
ways. Additionally, purified antigens (Hakomori, Ann. Rev. Immunol.
2:103, 1984) or anti-idiotypic antibodies (Nepom et al., Proc. Natl.
Acad. Sci. USA 81: 2864, 1985; Koprowski et al., Proc. Natl. Acad. Sci.
USA 81: 216, 1984) relating to such antigens could be used to induce an
active immune response in human patients.

[0311] The antibody compositions used are formulated and dosages
established in a fashion consistent with good medical practice taking
into account the condition or disorder to be treated, the condition of
the individual patient, the site of delivery of the composition, the
method of administration, and other factors known to practitioners. The
antibody compositions are prepared for administration according to the
description of preparation of polypeptides for administration, infra.

[0312] As is well understood in the art, biospecific capture reagents
include antibodies, binding fragments of antibodies which bind to
activated integrin receptors on metastatic cells (e.g., single chain
antibodies, Fab' fragments, F(ab)'2 fragments, and scFv proteins and
affibodies (Affibody, Teknikringen 30, floor 6, Box 700 04, Stockholm
SE-10044, Sweden; See U.S. Pat. No. 5,831,012, incorporated herein by
reference in its entirety and for all purposes)). Depending on intended
use, they also can include receptors and other proteins that specifically
bind another biomolecule.

[0313] The hybrid antibodies and hybrid antibody fragments include
complete antibody molecules having full length heavy and light chains, or
any fragment thereof, such as Fab, Fab', F(ab')2, Fd, scFv, antibody
light chains and antibody heavy chains. Chimeric antibodies which have
variable regions as described herein and constant regions from various
species are also suitable. See for example, U.S. Application No.
20030022244.

[0314] Initially, a predetermined target object is chosen to which an
antibody can be raised. Techniques for generating monoclonal antibodies
directed to target objects are well known to those skilled in the art.
Examples of such techniques include, but are not limited to, those
involving display libraries, xeno or humab mice, hybridomas, and the
like. Target objects include any substance which is capable of exhibiting
antigenicity and are usually proteins or protein polysaccharides.
Examples include receptors, enzymes, hormones, growth factors, peptides
and the like. It should be understood that not only are naturally
occurring antibodies suitable for use in accordance with the present
disclosure, but engineered antibodies and antibody fragments which are
directed to a predetermined object are also suitable.

[0315] The present disclosure also encompasses a kit comprising the
compounds of the invention or assay components of the invention and an
instructional material that describes administration of the compounds or
the assay. In another embodiment, this kit comprises a (preferably
sterile) solvent suitable for dissolving or suspending the composition of
the invention prior to administering the compound to the mammal.

[0316] Various aspects and embodiments of the invention are described in
further detail below.

EXAMPLE 1

[0317] Chemokines CXCL9, CXCL10, and CXCL11 Antimicrobial Activity

[0318] We tested whether human CXCL9, CXCL10, and CXCL11 exhibited
antimicrobial activity against B. anthracis. These interferon-inducible
(ELR-) CXC chemokines exhibited not only antimicrobial activity against
the vegetative form of the organism, but also the spore form such that
spore germination was blocked or reduced. An effect on spores is
unprecedented, even for any of the traditional antibiotics. We found a
hierarchy of activity with human CXCL10>CXCL9>CXCL11 in their
ability to kill bacilli and block spore germination. We also tested the
effects of recombinant murine CXCL9, CXCL10, and CXCL11 and found similar
effects but with a different hierarchy of activity:
CXCL9>CXCL10>CXCL11 (of note, human CXCL10 and murine CXCL9 exhibit
very similar antimicrobial and anti-spore effects at the same
concentrations).

[0319] Unless otherwise stated, recombinant human Interferon-inducible
(ELR-) CXC chemokines were used for the in vitro studies herein. As
controls, we used two recombinant human or mouse C-C family chemokines
(CCL2 and CCL5) that have a similar molecular mass and charge
(isoelectric point) as CXCL9, CXCL10, and CXCL11, but had no
antimicrobial activity against B. anthracis spores or bacilli. The
initial concentration of the interferon-inducible (ELR-) CXC chemokines
used in our in vitro studies was 48 ug/ml. The 50% effective
concentration (EC50) is 4-6 ug/ml for human CXCL10 or murine CXCL9, based
on concentration curves using 0-72 ug/ml of interferon-inducible (ELR-)
CXC chemokine. Although these concentrations may seem high based on the
recognized potency of these interferon-inducible (ELR-) CXC chemokines as
chemoattractants for recruitment of cells from distant locations, the
local concentrations generated by and around cells in the lungs are
likely higher. In this vein, these concentrations are commensurate with
concentrations recovered from nasal secretions and stimulated by
interferon-y in cell culture.

[0320] Immunogold electron microscopy (EM) studies of spores treated with
CXCL10 demonstrated that CXCL10 localized internal to (not outside) the
protective exosporium layer of the spores. In vegetative cells, CXCL10
localized primarily to the cell membrane (FIG. 2). These findings suggest
that interaction of these interferon-inducible (ELR-) CXC chemokines with
B. anthracis spores and vegetative cells is not simply due to a
charge-charge interaction with random distribution at the outer surface
of the organisms. Preliminary studies performed with stationary phase
vegetative bacilli revealed >10-fold more potent CXCL10 killing effect
with an EC50 value of 0.33 μg/m1 (FIG. 11C), compared to our
previously reported EC50 value of 4-6 μg/ml for CXCL10 against
exponential phase organisms.

[0321] These findings suggest that the interaction of these
interferon-inducible (ELR-) CXC chemokines with B. anthracis spores and
bacilli is not simply due to a charge-charge interaction with random
distribution at the outer surface of the organisms.

EXAMPLE 2

[0322] In vivo Activity of CXCL9, CXCL10, and CXCL11

[0323] To test the biological relevance of CXCL9, CXCL10, and CXCL11 in
vivo, we initially conducted a study comparing susceptible A/J and
resistant C57BL/6 mice inoculated with B. anthracis Sterne strain spores
that luminesce when undergoing germination. Using an in vivo Imaging
System (IVIS), spore germination was monitored over time after intranasal
inoculation of spores; little to no spore germination occurred in the
lungs of the resistant C57BL/6 mice while highly detectable levels of
germination were detected in the lungs of the A/J mice. Measurement of
CXCL9, CXCL10, and CXCL11 levels in lung homogenates from these animals
revealed that C57BL/6 mice had significantly higher levels of CXCL9 and
CXCL10 after spore inoculation than did A/J mice. In vivo neutralization
studies to further test the biological significance of these
interferon-inducible (ELR-) CXC chemokines revealed (FIG. 9) that
antibody neutralization of CXCL9, CXCL9/ CXCL10, or CXCL9/CXCL10/CXCL11,
but not CXCR3, rendered the C57BL/6 mice significantly more susceptible
to B. anthracis Sterne strain infection than the serum control-treated
animals. We obtained similar data using CXCR3 knockout mice as well.
These data support that there is a direct antimicrobial effect of these
interferon-inducible (ELR-) CXC chemokines in vivo as well as in vitro.
Furthermore the antimicrobial activity of both human and murine CXCL9,
CXCL10, CXCL11 has been established using physiological salt
concentrations against B. anthracis Sterne strain spores and bacilli.

[0324] Our observation that these interferon-inducible (ELR-) CXC
chemokines have antimicrobial activities against spores and bacilli is
strikingly novel and opens up an exciting avenue of research for studying
host interferon-inducible (ELR-) CXC chemokines as direct antimicrobial
agents and for developing novel therapeutic strategies using these
interferon-inducible (ELR-) CXC chemokines. To begin to determine the
mechanism of action of these interferon-inducible (ELR-) CXC chemokines
against this bacterial pathogen, we have used a highly innovative genetic
screening approach and identified a putative bacterial target of CXCL10;
this target is annotated in the B. anthracis genome as FtsX, the permease
component of an ATP-binding cassette (ABC) transporter that is widely
conserved among Gram-positive and Gram-negative bacterial species. The
identification of a putative bacterial target opens up exciting
possibilities for novel therapeutic targets using the
interferon-inducible (ELR-) CXC chemokines

EXAMPLE 3

[0325] Determination that FtsX is the Target for CXCL9, CXCL10, and CXCL11

[0334] The interferon-inducible (ELR-) CXC chemokines, CXCL9, CXCL10 and
CXCL11, are important components of host defense in a variety of
infections. We now have evidence that interferon-inducible (ELR-) CXC
chemokines have direct in vitro antimicrobial activity against B.
anthracis spores and bacilli.

[0336] 2) By immunogold EM imaging, CXCL10 localizes to spore structures
within and internal to the exosporium, namely, to the spore coat and
spore cortex; in vegetative cells, CXCL10 localizes to the cell membrane
(FIG. 2).

[0337] 3) CXCL10 exhibits direct antimicrobial activity against spores and
encapsulated cells of B. anthracis Ames strain (FIG. 3A) and against B.
anthracis Sterne strain (FIGS. 3B & 3C). These data indicate that the CXC
chemokines have antimicrobial effects against both unencapsulated and
encapsulated strains of B. anthracis and support the use of Sterne strain
as a model organism for the proposed studies.

[0338] 4) Initial screen of a B. anthracis Sterne strain transposon
mutagenesis library using CXCL10 yielded a number of resistant bacterial
isolates that are clones--the disrupted gene is annotated as ftsX and
encodes the permease component of a prokaryotic ABC transporter (FIG. 4).

[0339] Identification of a putative bacterial target of CXCL10. We used an
innovative genetic approach to identify a chemokine target in B.
anthracis bacilli. This approach entailed use of a mariner-based
transposon mutagenesis library adapted for B. anthracis Sterne strain
from Listeria monocytogenes and developed by investigators at University
of California, Berkeley (Zemansky, (2009) J. Bacteriol. 191:3950-3964).
The transposon randomly inserts into the chromosomal and plasmid DNA and
is designed to allow sequencing of regions flanking the transposon
insertion, thus enabling rapid identification of the disrupted gene. We
screened the B. anthracis transposon mutagenesis library for mutants that
were resistant (or less susceptible) to CXCL10 and identified eighteen
bacterial isolates (TNX1-18) resistant to CXCL10 in two independent
screens; 10 of these 18 isolates were confirmed to be resistant to CXCL10
using an Alamar Blue viability assay (FIG. 4). In multiple isolates, the
disrupted gene was identified by PCR and DNA sequencing as BAS5033,
annotated as ftsX. This gene has a high degree of homology to the gene
that encodes the Bacillus subtilis FtsX, an integral membrane protein
component of an ABC transporter (FIG. 5) that functions by importing
signals involved in the initiation of sporulation. This finding raises
intriguing questions about whether the B. anthracis homologue of FtsX
plays a role in transporting components/nutrients related to the
maintenance of viability and is a direct (or indirect) target of CXCL10,
or alternatively is involved in the uptake of CXCL10 into the organism. A
predicted topology of the B. anthracis FtsX is shown in FIG. 6.

[0340] We successfully created a knockout mutant of ftsX by bacteriophage
transduction (designated as the "ftsX mutant" or ΔftsX mutant or
ΔftsX) using published protocols (43) and confirmed resistance of
this mutant to CXCL10 (FIG. 7 and FIG. 12B). Furthermore, we have found
that this mutant strain is also resistant to CXCL9 and CXCL11 (FIG. 8),
which supports our hypothesis that CXCL9, CXCL10, and CXCL11 have a
common target in vegetative bacteria.

[0341] Generation of a clean deletion mutant (designated "ΔftsX")
will allow for gene complementation analysis to verify that the original
(susceptible) phenotype is restored. Once validated, the ΔftsX
strain will be tested in vitro for its resistance to various
concentrations CXCL9, CXCL10, and/or CXCL11. EM will be used to assess
the structural integrity of the 4ftsX bacilli treated with CXCL9, CXCL10,
or CXCL11, and immunogold EM will be used to assess interferon-inducible
(ELR-) CXC chemokine localization in ΔftsX bacilli compared to that
in wildtype Sterne 7702 bacilli.

[0342] Co-localization studies. To address the hypothesis that CXCL10
interacts directly with FtsX rather than indirectly (by affecting a
molecule that interacts with FtsX), we will take the following approach.
Because there are no FtsX-specific antibodies available at this time, we
will generate a tagged version of the B. anthracis permease. In
collaboration with Dr. Stibitz, we plan to generate an FtsX-GFP fusion
protein with the GFP located at the carboxyl terminal end of FtsX using
allelic exchange (see reference 61) methodology previously employed in B.
anthracis. Our choice of GFP is based on published studies of GFP fusion
proteins in B. anthracis and successful expression and use of FtsX-GFP
fusion proteins generated in B. subtilis and other bacterial species.
Advantages to using a GFP tag are that we will be able to monitor the
location and expression of FtsX at various stages of growth during the
experiments, which may prove important if B. anthracis is killed by
CXCL10 by, for example, disruption of cell division by inhibiting septal
ring formation (FtsX localizes to septal rings in B. subtilis (34a)).
Importantly, the tag introduced into FtsX must not interfere with the
function of the transporter. Since the substrate transported by FtsX is
unknown, we will test for potential disruption of FtsX function by
assessing bacterial growth in medium alone (no chemokine), monitor
kinetics of cell division in log phase, monitor formation of septal rings
using a GFP tagged version of FtsX under fluorescence microscopy as per
published protocols (see references 28, 41), and assess the ability of
bacilli to form spores (since FtsX in B. subtilis is thought to play a
critical role in sporulation). We will perform EM to assess the
structural integrity of the bacteria expressing untagged versus tagged
FtsX at various stages of vegetative growth and sporulation.

[0343] Once a B. anthracis strain with a GFP-tagged FtsX is created and
tested, we will examine the susceptibility of the organism to CXCL10 to
ensure that addition of the tag has not altered the antimicrobial effect
of CXCL10 against the bacilli. We will then perform co-localization
studies with CXCL10 using immunofluorescence/confocal microscopy to study
the interaction of CXCL10 with FtsX at various time points. B. anthracis
cells that produce FtsX-GFP will be fixed and permeabilized using
standard protocols familiar to the PI (58) and tested to ensure that the
fixation process did not reduce or quench the GFP signal. If this does
occur, an alternative approach would be to add anti-GFP antibodies after
the bacteria are fixed and permeabilized followed by fluorescent-labeled
secondary antibody. Anti-CXCL10 antibodies will be used followed by a
(red) fluorescent-labeled secondary antibody.

[0344] Immunofluorescence/confocal microscopy will be performed to
determine the individual locations of the CXCL10 and FtsX in the bacteria
and if there is co-localization by appearance of a yellow signal (overlap
of red and green signals). Similar studies will be performed using CXCL9
and CXCL11.

[0345] Site-directed mutagenesis studies. Without wishing to be bound by
any particular theory, we hypothesize that CXCL10 (as well as CXCL9 and
CXCL11) interacts with the predicted extracellular portions of FtsX,
designated as Loops 1 and 2 in FIG. 6. To assess which portions of FtsX
may interact with CXCL10, we will use allelic exchange to create deletion
mutants of segments of Loop 1 and Loop 2. Depending on results obtained
with mutants of Loop 1 and Loop 2, additional mutants of Loop 3 and 4
(predicted intracellular loops) will be generated. If deletion of a
segment of FtsX abrogates the CXCL10 effect on the bacilli, we will
narrow our studies to focus on key amino acids responsible for the
interaction and/or effect of CXCL10; to do this, we will perform
site-directed mutagenesis with substitution of neutral amino acids
(alanine) for select amino acids in the portion of FtsX that may be
responsible for the interaction or effect of CXCL10. We will initially
target negatively charged amino acids that are clustered together since
the net charge distribution is likely to play an important role in the
interaction with CXCL10, which has a positively charged carboxyl
terminus. The ability of the site-directed mutagenesis to disrupt
interactions between the interferon-inducible (ELR-) CXC chemokine and
FtsX will be assessed by in vitro susceptibility testing of the mutant
bacterial strain to the interferon-inducible (ELR-) CXC chemokine
Further, using a GFP-tagged version of FtsX, we will perform: 1)
co-localization studies with immunofluorescence microscopy; and 2)
immunoprecipitation coupled with Western blot analyses to determine if
the mutated FtsX can be co-precipitated with antibodies to the specific
interferon-inducible (ELR-) CXC chemokine

[0346] Expected results and interpretations. We expect that FtsX is the
target for CXCL9, CXCL10, and CXCL11. Furthermore, we anticipate that the
interaction between chemokine and FtsX is a direct interaction at the
extracellular portion of the permease at a location where there is a net
negative charge distribution. We anticipate that co-localization
immunofluorescence experiments will reveal that the proteins interact at
the cell membrane. Further, we predict that performing site-directed
mutagenesis of select extracellular portions and then select (negatively
charged) amino acids will abrogate the interaction and the antimicrobial
effect of the interferon-inducible (ELR-) CXC chemokine against the
bacilli.

[0347] Future extensions of these studies. In the studies described above,
our focus has been on studying the interaction of the
interferon-inducible (ELR-) CXC chemokines with vegetative bacilli with
primary attention to the role of FtsX as a putative target. This
represents the first description of the direct antimicrobial activity of
interferon-inducible (ELR-) CXC chemokines against spores. This finding
opens up the possibility of developing anti-spore therapeutics that could
be used as an adjunct to conventional antimicrobials that only act
against the vegetative form of the organism. Based on the structural
differences and metabolic activities of these two different forms of the
same organism, we hypothesize a priori that the interferon-inducible
(ELR-) CXC chemokine targets and mechanisms of action differ between
spores and bacilli. In fact, preliminary testing of spores produced by
the ftsX mutant strain were not resistant to CXCL10. We propose as a
future extension of our studies to pursue identification of spore targets
of the interferon-inducible (ELR-) CXC chemokines The approach will
entail screening of B. anthracis spores derived from sporulation of the
vegetative transposon mutagenesis library.

[0348] Timing of chemokine-spore interaction will require careful
monitoring since any spores resistant to the chemokine will germinate
under germination-permissive conditions. Since there is no assurance that
the resultant bacilli will be resistant to the chemokine present in the
medium, the bacilli will likely be killed and not be identified as an
interferon-inducible (ELR-) CXC chemokine resistant spore isolate. An
alternative approach will be to incubate spores with the
interferon-inducible (ELR-) CXC chemokine under germination
non-permissive conditions (i.e., water or medium with no serum) for the
minimal time of 60 minutes required for interferon-inducible (ELR-) CXC
chemokine exposure to elicit an antimicrobial effect on the spores, based
on washout experiments and then place the spores in germination
permissive medium without interferon-inducible (ELR-) CXC chemokine.
Bacilli derived from the chemokine-resistant spores will be isolated for
further analysis and identification of mutant gene(s).

EXAMPLE 4

[0349] Utilizing the interferon-inducible interferon-inducible (ELR-) CXC
chemokines to elicit a protective effect in vivo against pulmonary
anthrax infection in a mouse model.

[0350] The data described herein support the notion that the
interferon-inducible interferon-inducible (ELR-) CXC chemokines play a
direct and critical role in protecting the host against pulmonary
anthrax. In addition to the in vitro data already presented in Examples
1-3, in vivo data provide further support as follows:

[0351] 1) IFN-γ, CXCL9, CXCL10, CXCL11 are markedly induced and
expressed early in the lungs of C57BL/6 mice, which are highly resistant
to inhalational spore challenge.

[0354] Neutralization of CXCL9, CXCL9/CXCL10, or CXCL9/CXCL10/CXCL11 but
not CXCR3 renders C57BL/6 mice susceptible to B. anthracis spore
challenge. To assess the biological role of CXCL9, CXCL10, CXCL11, or
their shared CXCR3 receptor (which is expressed by leukocytes recruited
by CXCL9-11), we performed a survival study using C57BL/6 mice that
received intraperitoneal (i.p.) injections of control serum or
anti-CXCL9, anti-CXCL10, anti-CXCL11, anti-CXCL9+anti-CXCL10,
anti-CXCL9+anti-CXCL10+anti-CXCL11, or anti-CXCR3 serum 24 hr prior to
intranasal spore challenge and then daily throughout the experiment,
using published protocols (see references 12, 13, 65, 108). The
anti-CXCL9, CXCL10, and CXCL11 neutralizing antibodies have been
validated in published work (see references 12, 19, 108). As shown in
FIG. 9, mice that received anti-CXCL9, anti-CXCL9+anti-CXCL10, or
anti-CXCL9+anti-CXCL10+anti-CXCL11 had significantly decreased survival
after spore challenge. The other groups, including animals that received
anti-CXCR3, had no significant difference in survival compared to normal
serum controls that received spore challenge. These findings suggest that
CXCL9, CXCL10, CXCL11 have significant direct antimicrobial effects
against B. anthracis in vivo that may be independent of cell recruitment
of CXCR3-expressing cells.

[0355] Determining that FtsX is a target of CXCL9, CXCL10, and CXCL11
using an in vivo model of infection.

[0356] Both wildtype B. anthracis and the ΔftsX chemokine-resistant
mutant will be used in a mouse model of pulmonary infection to determine
whether FtsX is a target for CXCL9, CXCL10, and CXCL11, leading to a
protective antimicrobial effect in vivo. C57BL/6 mice are resistant to
pulmonary infection with B. anthracis Sterne strain, but are susceptible
to B. anthracis introduced by other routes of inoculation (e.g.,
subcutaneous). In contrast, A/J mice are highly susceptible to B.
anthracis Sterne strain infection introduced via any of the above routes
of inoculation. Thus, it would appear that C57BL/6 mice have an effective
pulmonary host defense response/mechanism that is present or is generated
in the lungs of mice infected with this pathogen. We previously found
that lungs from C57BL/6 mice had significantly higher levels of CXCL9 and
CXCL10 induced after intranasal inoculation of spores than did those from
A/J mice. As noted above, we have also observed that neutralization of
CXCL9, CXCL9/CXCL10, or CXCL9/CXCL10/CXCL11 rendered C57BL/7 mice
susceptible to an inhalational disease (FIG. 9). Using the two mouse
strains and a chemokine-resistant B. anthracis ΔftsX strain, we
will further investigate the role of the interferon-inducible (ELR-) CXC
chemokines during lung infection.

[0357] Without wishing to be bound by any particular theory, it is
hypothesized herein that CXCL9, CXCL10, and CXCL11 have a direct
antimicrobial effect both in vitro and in vivo against B. anthracis via
FtsX such that absence of FtsX will render resistant mice susceptible to
infection. We will determine whether the absence of FtsX causes normally
resistant C57BL/6 mice to become susceptible to pulmonary infection. The
study groups will be: 1) C57BL/6 mice+intranasal B. anthracis Sterne
strain (parent strain) spores; 2) C57BL/6 mice+intranasal B. anthracis
ΔftsX spores; and 3) C57BL/6 mice+intranasal B. anthracis Sterne
strain (parent strain) spores+anti-CXCL9/CXCL10/CXCL11 neutralizing
antibodies.

[0358] Mouse survival will be followed over a 20-day period following
spore challenge. A minimum of 10 animals per group×3 groups=30 mice
will be required for survival studies. We will assess burden of infection
caused by the wildtype and the ΔftsX strain of B. anthracis by
determining bacterial colony forming units (CFUs) and histopathology in
the lungs as the initial site of infection and in the kidneys as a
measure of bacterial dissemination to other organs. Using CFU data in
conjunction with histopathology, we will determine whether there is more
severe localized lung infection and/or if there is increased
dissemination of bacteria to distal organs as a consequence of the
absence of FtsX. The lungs and kidneys from animals will be harvested at
an early and a later time point (e.g., day 2 and day 7 post-infection)
for determination of bacterial CFUs from tissue samples (+/- heat
treatment since spores are heat resistant whereas vegetative bacilli are
heat sensitive) plated on BHI agar plates and incubated overnight at
37° C. In these studies, a minimum of three mice per group will be
required per time point for these determinations (i.e., three mice per
group per time point×3 groups×2 time points=18 mice). We will
collect tissues (lungs, mediastinal lymph nodes, spleen, kidneys, liver)
for histopathology to assess tissue damage, infiltration of leukocytes
into the tissues, and spore/bacilli burden and localization/distribution
within the tissues.

[0359] The samples will be reviewed and graded for the level of
inflammation using the same severity scale as previously described (see
references 12, 13, 108). The tissues will also be stained and examined
for spores and bacilli, using published protocols (see reference 94). A
minimum of 3 animals per group will be needed for histopathology=3 mice
per group×3 groups×2 time points=18 mice. Thus, a total of
30+18+18=66 mice will be needed for these studies. For 3 replicate
experiments, a total of 66×3=198 mice total will be required.
Statistical analyses will be used to compare the data from each group to
their respective control groups as well as between treatment groups.

[0360] Expected results and interpretations: Our data (FIG. 9) support the
hypothesis that CXCL9, CXCL10, and CXCL11 are involved in the resistance
of C57BL/6 mice to intrapulmonary B. anthracis infection such that
neutralization of CXCL9, CXCL9/CXCL10, or CXCL9/CXCL10/CXCL11 renders
C57BL/6 mice susceptible to pulmonary anthrax infection (FIG. 9).
Furthermore, we have data showing that CXCL10-/- mice have increased
spore/vegetative bacilli CFUs after spore challenge compared to that of
the C57BL/6 parent strain. We expect that, in contrast to exhibiting
resistance to wildtype B. anthracis, C57BL/6 mice inoculated with B.
anthracis ΔftsX will succumb to infection with dissemination and
mortality rates similar to or higher than the C57BL/6 mice inoculated
with wildtype B. anthracis+anti-CXCL9/CXCL10/CXCL11 neutralizing serum.

[0361] Determine that interferons promote host defense against B.
anthracis infection through the induction of CXCL9, CXCL10, and CXCL11.

[0362] Our data support that CXCL9, CXCL10, CXCL11, and IFN-γ are
generated in the lungs as early as 1-6 hours after spore challenge;
however, type 1 interferons were not measured in those experiments. We
will determine which interferons are primarily responsible for inducing
CXCL9, CXCL10, CXCL11 after spore challenge to determine how chemokines,
as potential therapeutics, could be induced after a host has acquired the
infection. Our working hypothesis is that type 1 and type 2 interferons
are responsible for inducing these interferon-inducible (ELR-) CXC
chemokines during anthrax infection.

[0363] Initially, we will perform intranasal spore challenges of IFN-y
receptor knockout (IFN-γR KO) mice (Jackson Labs), using the
C57BL/6 parent strain as a control. Lungs will be harvested at 1, 6, 24,
48 hrs post-infection (same time points as in ref. 26) for: a) CFU
determinations and b) ELISAs to measure CXCL9, CXCL10, CXCL11 levels in
lung homogenates. CFU determination will be performed using heated and
unheated aliquots to assess spore CFUs (i.e., from heated samples) and
the total number of spore+bacilli CFUs (i.e., from unheated samples). A
minimum of three mice per group will be needed for tissue CFU
determination and chemokine quantification at each of the four time
points. Thus, a minimum of 3 mice per group×2 groups×4 time
points=24 mice. After these fundamental data are obtained, mouse survival
will be monitored over a 20-day period. A minimum of 10 animals per
group×2 groups=20 mice for survival studies. Therefore, a total of
24+20=44 mice×3 replicate experiments=132 mice will be needed for
these experiments. Statistical analyses will be used to compare data from
each group to their respective control groups and between treatment
groups.

[0364] We anticipate that the IFN-γR KO mice will exhibit markedly
increased susceptibility to B. anthracis challenge. We predict that the
levels of CXCL9, CXCL10, and CXCL11 in lung homogenates will be low
compared to the C57BL/6 parent strain and that CFUs will be higher in the
IFN-γR KO mice. These results would support our hypothesis that
IFN-γ is key in inducing the interferon-inducible (ELR-) CXC
chemokines during B. anthracis infection. If we find opposite results
(i.e., that the IFN-γR KO mice remain resistant like the C57BL/6
parent strain), then it is likely that the type 1 interferons play a key
role.

[0365] Develop a therapeutic strategy in a pre-clinical animal model with
interferon induction of CXCL9, CXCL10, and CXCL11 to treat bacterial
infections.

[0366] A pre-clinical animal model will be used to test the hypothesis
that interferon-inducible (ELR-) CXC chemokines can function as
therapeutics. Since CXCL9, CXCL10, and CXCL11 are potently induced by
type 1 and type 2 interferons, we will focus on testing the utility of
administering exogenous interferons as a therapeutic strategy for B.
anthracis infection. A major advantage to the use of exogenous
interferons is that type 1 interferons (IFN-α/β) and type 2
interferon (IFN-γ) are well-studied, FDA-approved drugs for human
use, primarily for infectious diseases such as viral infections (type 1
interferons) and mycobacterial diseases (IFN-γ). Thus, there is a
track record for clinical use of these immunomodulatory agents that we
can draw upon for our proposed experiments. Especially pertinent to this
proposal is the precedent in the literature that IFN-β or the Type 1
inducer (poly-ICLC) confers protection in mice infected with B. anthracis
Ames strain (see reference 107).

[0367] In a pilot experiment, we injected A/J mice with recombinant murine
IFN-γ (20,000 units i.p.), collected lungs at 0, 1, 6, 18, and 24
hrs for homogenization, and measured the levels of CXCL9, CXCL10, and
CXCL11 in the homogenates by ELISA. We found that CXCL9, CXCL10, and
CXCL11 levels peaked at 6 hours with levels of 6645±1399 pg/ml,
5503±1022 pg/ml, and 1631±356 pg/ml, respectively; these
concentrations were within the range of the levels we previously observed
in our studies of resistant C57BL/6 mice. Animals that were monitored for
72 hours (endpoint of the experiment) after IFN-γ administration
remained healthy. Thus, we can induce CXCL9, CXCL10, and CXCL11 in the
lungs using exogenous IFN-γ. We hypothesize that the results of
administration of IFN-γ in a susceptible mouse strain will lead to
the development of novel immunomodulatory approaches for post-exposure
prophylaxis or treatment of anthrax.

[0368] We will test the effectiveness of administration of exogenous
interferons, focusing initially on IFN-γ, as an immunomodulatory
agent for treating B. anthracis pulmonary infection. Our pilot studies
noted above with IFN-γ used an i.p. route of administration, and we
will initially plan to administer via the i.p. route for survival studies
since Walberg et al. found that i.p. administration of exogenous
IFN-β provided greater protection that by the intranasal route (see
reference 107). One caveat is that the same group found that
administration of the type 1 inducer (poly-ICLC) via intranasal route was
more protective than via the i.p. route, so the route of administration
is an important variable that may require further testing. The arms of
the study will be: 1) sham-infected mice+IFN-γ (as a control to
ensure that IFN-γ is not contributing to morbidity/mortality of the
mice); 2) spore-infected mice without IFN-γ (as a control to ensure
that spore challenge worked); 3) spore-infected A/J mice +IFN-γ;
and 4) spore-infected A/J mice+IFN-γ+anti-CXCL9/CXCL10/ CXCL11
neutralizing Abs (to test whether a protective effect conferred by
IFN-γ is due to the production of CXCL9, CXCL10, and CXCL11).
Measurements will include: 1) host survival (monitored for 10-15 days);
2) CXCL9, CXCL10, and CXCL11 levels in the lungs of animals at days 2, 5,
and 10 after spore challenge; 3) Lung and kidney bacterial CFU
determination to assess localized burden of infection as well as
dissemination of organisms to other organs; 4) histopathology to assess
tissue damage in the lungs. A minimum of 10 animals per group×4
groups=40 animals will be needed for survival studies. A minimum of 3
animals per group×4 groups will be needed for CFU determination,
histopathology, and interferon-inducible (ELR-) CXC chemokine measurement
by ELISA=12×4 outcome measurements=48 animals. Thus, a total of
40+48 mice=88 mice×3 replicates=264 mice will be needed.

[0369] With IFN-γ treatment, we anticipate that the A/J mice will
have improved survival after spore challenge. In contrast, we anticipate
that administration of IFN-γ plus neutralizing Abs against
CXCL9/CXCL10/CXCL11 will result in the mice being highly susceptible to
anthrax infection as seen with spore-challenged control A/J mice.

[0370] Walberg et al. (see reference 107) found that IFN-⊖ or the
type 1 inducer poly ICLC conferred a protective effect for Swiss Webster
mice infected with B. anthracis Ames strain. Mouse strain, choice of
interferon, and dose/route of interferon administration are all potential
variables. By measuring CXCL9, CXCL10, and CXCL11 levels generated in the
lungs and assessing histopathology at various time points after spore
challenge and while the animals are receiving IFN-γ will help us
assess the appropriateness or potentially, the under- or
over-responsiveness of the host response. It is also possible that type I
interferons (e.g., IFN-α/β) or a combination of
IFN-α/β and IFN-γ are the more relevant interferons (or
interferon combinations) for inducing a protective effect in our
pre-clinical model.

[0371] The in vitro and in vivo experimental approaches and translational
nature of the proposed project will allow extensive characterization of a
novel antimicrobial effect whereby CXCL9, CXCL10, and CXCL11 produced in
the lungs have direct antimicrobial effects against B. anthracis spores
and bacilli. We recently identified a putative bacterial target from a B.
anthracis transposon mutagenesis library screen; the finding of FtsX as a
target of a chemokine (directly or indirectly) is an entirely novel
finding that opens up exciting avenues of investigation that should lead
to innovative therapeutic strategies for treating and/or preventing
pulmonary anthrax. These findings will likely extend beyond B. anthracis
and have therapeutic impact on infections caused by a range of pathogenic
and potentially, multi-drug resistant bacteria.

[0373] CXCL10 has been found to exert a markedly more potent effect
against stationary phase B. anthracis Sterne strain 7702 (wildtype)
organisms (see FIGS. 11A-B). Overnight cultures were either diluted back
in fresh medium and grown to exponential phase prior to addition of
buffer control or CXCL10 at 8 μg/ml (ie, ˜EC50 value, see
FIG. 11A) or used directly from overnight cultures by spinning down,
reconstituting in same volume fresh medium plus buffer control or CXCL10
at 8 μg/ml (FIG. 11B). Aliquots were plated out for CFU determination
after an incubation of 30 min or 1 hr. A concentration curve for CXCL10
against stationary phase organisms is shown in (FIG. 11C) with an
EC50 value determined to be 0.33+/-0.05 μg/ml. Each experiment
was performed 3 separate times in triplicates. n.d., not detected.

[0374] The EC50 value (0.33+/-0.05 μg/ml) for CXCL10 (FIG. 11C) is
>10-fold more potent against stationary phase organisms compared to
the EC50 value determined for exponential phase organisms (as shown
in FIG. 12B for the wildtype B. anthracis Sterne strain designated "7702
wt" in the graph). Importantly, the stationary phase organisms were
placed in fresh culture medium at the time of the assay with CXCL10 so
that, for the short assay incubation period of 30-60 minutes, there are
nutrients present. Since the assay medium is not nutrient depleted, the
finding that CXCL10 is more effective appears to not be simply due to a
lack of nutrients for the organisms making them less fit or a lack of a
nutrient or other component in the medium that could otherwise compete
with CXCL10 for targeting FtsX or other target.

EXAMPLE 6

[0375] Generation of a B. anthracis ΔftsX mutant strain.

[0376] Markerless allelic exchange was used to create a deletion mutant of
the ftsX gene in wildtype B. anthracis Sterne strain (designated
"ΔftsX"), using protocols of Dr. Stibitz (see references 30, 63,
76). Growth characteristics are shown in FIG. 12A for wildtype B.
anthracis Sterne strain 7702 and ΔftsX. The ΔftsX strain
grows more slowly than wildtype strain. The ΔftsX strain has a
distinctive phenotype such that bacilli grow in "kinked" chains due to
various angles produced at septations between individual bacilli.
Sporulation occurs with ΔftsX but with a lower yield than that of
the parent strain. We confirmed resistance of ΔftsX to CXCL10 (FIG.
12B). Furthermore, we found that ΔftsX was also resistant to CXCL9
and CXCL11, which supports that CXCL9, CXCL10, and CXCL11 have a common
target in vegetative bacteria. Additionally, in contrast to B. anthracis
Sterne 7702 strain, the ΔftsX exponential and stationary phase
organisms are both resistant to CXCL10.

EXAMPLE 7

[0377] Determining the Localization of CXCL10 in the Bacterial Cells
Relative to FtsX

[0378] Co-localization studies. To assess localization of CXCL10 in the
bacterial cells and whether it interacts directly with FtsX, we will take
the following approach. Since there are no FtsX-specific antibodies
available at this time, we will generate a tagged version of the B.
anthracis FtsX. We plan to generate an FtsX-GFP fusion protein with the
GFP located at the C-terminal end of FtsX using allelic exchange
methodology previously employed in B. anthracis. Our choice of GFP is
based on published studies of GFP fusion proteins in B. anthracis and
successful expression and use of FtsX-GFP fusion proteins generated in E.
coli, B. subtilis, and other bacterial species (see references 7, 31, 49,
97). Advantages to using a GFP tag are that we will be able to monitor
the location and expression of FtsX at various stages of growth during
the experiments, which may prove important if B. anthracis is killed by
CXCL10 by, for example, disruption of cell division by inhibiting septal
ring formation (FtsX localizes to septal rings in E. coli and B.
subtilis).

[0379] Importantly, the tag introduced into FtsX must not interfere with
the function of the transporter. Since the substrate transported by FtsX
is unknown, we will test for potential disruption of FtsX function by
assessing bacterial growth in medium alone (no chemokine), monitor
kinetics of cell division in log phase, monitor formation of septal rings
using a GFP tagged version of FtsX under fluorescence microscopy as per
published protocols (see references 31, 49), and assess the ability of
bacilli to form spores (since FtsX in B. subtilis is thought to play a
critical role in sporulation). We will perform EM to assess the
structural integrity of the strain expressing tagged FtsX versus wildtype
strain at various stages of vegetative growth and sporulation.

[0380] Once a B. anthracis strain with a GFP-tagged FtsX is created and
tested, we will examine the susceptibility of the organism to CXCL10 to
ensure that addition of the GFP tag has not altered the antimicrobial
effect of CXCL10 against the bacilli. We will perform co-localization
studies with CXCL10 using immunofluorescence/ confocal microscopy to
study the interaction of CXCL10 with FtsX at various time points. B.
anthracis cells that produce FtsX-GFP will be fixed and permeabilized
using standard protocols (see reference 60) and tested to ensure that the
fixation process did not reduce or quench the GFP signal. If this does
occur, an alternative approach would be to add anti-GFP antibodies after
the bacteria are fixed and permeabilized followed by fluorescent-labeled
secondary antibody. Commercially available anti-CXCL10 Abs will be used
followed by a (red) fluorescent-labeled secondary antibody.
Immunofluorescence/confocal microscopy will be performed to determine the
individual locations of the CXCL10 and FtsX in the bacteria and determine
if there is co-localization by appearance of a yellow signal (overlap of
red and green signals).

[0381] E. coli reagents. Much of the work on FtsX and its related ABC
transporter components (FtsE, FtsY) has been performed in E. coli. An E.
coli ΔftsEX mutant strain (see reference 7) and plasmids for
complementation studies have been obtained from Dr. David Weiss
(University of Iowa). Additionally, E. coli strains that express
GFP-tagged FtsX or HA-tagged FtsE; plasmids for studying FtsY are also
available. Importantly, it has been published by others that CXCL10
exhibits antimicrobial effects against lab strains of E. coli (see
references 25, 112), and we have obtained data to support that E. coli
multi-drug resistant clinical isolates are susceptible to CXCL10
antimicrobial activity (see FIG. 13). Furthermore, testing of E. coli
ΔftsEX strain shows that this mutant strain exhibits increased
resistance to CXCL10 (FIG. 14), supporting that FtsX (or FtsEX) is
involved in susceptibility to CXCL10 in more than one bacterial species,
namely in E. coli as well as B. anthracis. Complementation studies with
plasmids encoding ftsX and/or ftsE are underway.

EXAMPLE 8

[0382] Determining the Role of the Other ABC Transporter Components, FtsE
and FtsY, in Susceptibility of Bacteria to CXCL10

[0383] Since ftsE is located immediately upstream of ftsX in the same
operon, and both gene products (FtsE and FtsX) play a linked and pivotal
role as the main components of the ABC transporter, we will investigate
whether ftsE impacts the susceptibility of the organism to CXCL10. An
important consideration is that FtsE is the ATP-binding component of the
ABC transporter and as such, deletion of it may tell us whether CXCL 10
is actively transported by FtsX or not. Therefore, we will delineate
whether CXCL10 (or a portion of it) is actively transported by FtsX or in
some way requires an active functioning transporter. On the other hand,
if deletion for ftsE has no impact on susceptibility to CXCL 10, then
active transport seems an unlikely requirement and would indicate that
CXCL10 requires FtsX for some other purpose in the cells. Although ftsY
is located elsewhere in the B. anthracis genome, its gene product, FtsY,
may play a role in the CXCL10 requirement for FtsX in order for it to
exert its antimicrobial activity. For this reason, we will investigate
this component of the ABC transporter as well.

[0384] The experimental approach will be similar to our approach for
generating the B. anthracis ΔftsX mutant strain described above. In
brief, we will use markerless allelic exchange to create a deletion
mutant of the ftsE or ftsY gene in wildtype B. anthracis Sterne strain
(designated ΔftsE or ΔftsY, respectively). Also, we will
generate a double deletion mutant of ftsE and ftsX (i.e., ΔftsEX)
in order to study the impact of the absence of these components of the
ABC transporter. Complementation studies will be performed using plasmids
with the genes for ftsE, ftsX, or ftsY, using plasmid constructs similar
to those already used and validated in B. anthracis. Controls for
complementation studies will include use of empty plasmid vectors as well
as non-transformed wildtype (parent) bacterial strains.

[0385] For each B. anthracis mutant strain created, we will test growth
characteristics and assess the viability of the organisms under various
growth conditions. We will draw upon the E. coli and B. subtilis
literature to assess particular growth requirements of the mutants such
as salt and sucrose to help maintain viability. Additionally, these may
be temperature sensitive mutants based on the literature, and temperature
requirements may need to be carefully assessed; for example, the E. coli
ΔftsEX mutant strain grows better at 30° C. and in the
presence of salt and sucrose for osmotic stabilization. Susceptibility
testing with CXCL10 will be performed using CFU determination or Alamar
Blue assay. Modifications to the assay will be guided by the information
gained from establishment of optimal growth conditions. To date, we have
used tissue culture medium with physiological salt concentrations as well
as other ions and proteins, so a requirement for salt and sucrose in the
assay medium is not anticipated to affect the CXCL10 susceptibility
assays that we routinely perform. We expect that, if CXCL10 requires the
active transport function of FtsX, then the ΔftsE mutant strain
should also be resistant to CXCL10-mediated killing as observed for
ΔftsX. If, however, CXCL10 (or a portion of it) does not require
the active transport by FtsX, then the ΔftsE mutant strain should
remain susceptible to CXCL10. I

[0386] Fractionation and Co-Immunoprecipitation Studies.

[0387] To further study localization of CXCL10 in vegetative cells, we
will perform fractionation studies using published protocols to separate
bacterial cell wall, membrane, and cytosolic fractions at various time
points using CXCL10-treated wildtype Sterne strain and a strain that
expresses tagged FtsX. We will assess localization of FtsX and CXCL10 in
the fractions using commercially available antibodies to the FtsX tag and
to CXCL10. Controls will include use of wildtype Sterne strain vs.
ΔftsX (the latter to compare the effect of the absence of FtsX on
CXCL10 localization) plus the B. anthracis strain that expresses tagged
FtsX. Gel electrophoresis followed by Western blot analysis will be used
to determine which fraction, if any, contains CXCL10. Fraction purity
will be determined by Western blot analysis in which the presence or
absence of Protective Antigen (a cytosolic/secreted protein) or the S
layer protein EA1 (which fractionates with the cell wall) is examined.

[0388] Co-immunoprecipitation studies using anti-CXCL10 antibodies will be
performed to test CXCL10 interaction with FtsX and/or identify other
interacting proteins. B. anthracis wildtype Sterne strain and ΔftsX
will be lysed by sonication, and aliquots of whole lysates (or
fractionated lysates) will be incubated with CXCL10 followed by
immunoprecipitation of CXCL10 and its interacting proteins using
commercially available anti-CXCL10 Abs and protein-G beads. Controls will
include buffer controls and appropriate isotype Ab controls. Gel
electrophoresis to separate proteins will be performed, followed by
silver staining for protein visualization; candidate bacterial targets
will be identified by mass spectrometry performed at our UVA Biomolecular
Research Core Facility.

[0389] We anticipate that fractionation studies will reveal that CXCL10
localizes to the cell membrane fraction and that co-immunoprecipitation
studies will reveal that CXCL10 interacts with FtsX. It is anticipated
that other proteins may be immunoprecipitated with CXCL10, and those
proteins deemed significant (i.e., not due to non-specific binding) will
be identified by mass spectrometry.

[0391] We hypothesize that CXCL10 interacts with the predicted
extracellular portions of FtsX, designated Loops 1 & 2 in FIG. 6, with
particular attention to Loop 1 based on the length of the loop, the
number of negatively charged amino acids, and the region of sequence
similarity to the CXCL10 receptor, CXCR3. Accordingly, we anticipate that
the interaction between CXCL10 and FtsX involves a direct interaction
with FtsX Loop 1 at a location where there is a net negative charge
distribution (more specifically, in the region of amino acids 54-80 with
similarity to the CXCR3 receptor binding region for CXCL10). We
anticipate that co-localization experiments will reveal that the proteins
interact at the cell membrane. We also predict that performing
site-directed mutagenesis of extracellular portions will abrogate the
interaction and the antimicrobial effect of CXCL10.

[0392] To assess which portions of FtsX interact with CXCL10, we will use
allelic exchange to create deletion mutants of segments of Loop 1 and
Loop 2. If deletion of a segment of FtsX abrogates the CXCL10 effect on
the bacilli, we will narrow our studies to focus on key amino acids
responsible for the interaction and/or effect of CXCL10. To do this, we
will perform site-directed mutagenesis with substitution of neutral amino
acids (alanine) for select amino acids in the portion of FtsX that may be
responsible for the interaction or effect of CXCL10. We will initially
target negatively charged amino acids that are clustered together since
the net charge distribution is likely to play an important role in the
interaction with CXCL10, which has a positively charged carboxyl
terminus. The ability of the site-directed mutagenesis to disrupt
interactions between the interferon-inducible (ELR-) CXC chemokine and
FtsX will be assessed by in vitro susceptibility testing of the mutant
bacterial strain to the interferon-inducible (ELR-) CXC chemokine Using a
GFP-tagged version of FtsX, we will perform: 1) co-localization studies
with immuno fluorescence microscopy; and 2) immunoprecipitation coupled
with Western blot analyses to determine if the mutated FtsX can be
co-precipitated with Abs to CXCL10.

Identifying the Region(s) of CXCL10 Responsible for its Antimicrobial
Effect

[0393] Identifying the region of CXCL10 responsible for its antimicrobial
activity is an important aspect that could lead not only to development
of a valuable tool for carrying out further mechanistic experiments but
also potentially lead to a therapeutic reagent for testing as an
antimicrobial agent. There are two very interesting and key
considerations: 1) CXC10 has highly positively charged C-terminus that
forms a predicted α-helix (FIG. 1) with similarity to defensins and
other cationic antimicrobial peptides; and 2) Sequence alignment of B.
anthracis FtsX and the known CXCL10 receptor, CXCR3, reveals that the two
proteins share ˜45% amino acid sequence similarity in one region of
the extracellular Loop 1 of each protein; the region in CXCR3 (amino
acids 9-35) includes a key domain for binding the N-terminal region of
CXCL10. We believe the C-terminal a-helical region of CXCL10 is
responsible for its direct antimicrobial activity while the N-terminal
portion of CXCL 10 may play a role in facilitating interaction with its
target, FtsX. This could potentially be somewhat analogous to
cholesterol-dependent cytolysins that bind to a cholesterol receptor and
insert a different portion of the molecule into the eukaryotic membrane
as an oligomer to form a pore, causing cell death.

[0394] Testing CXCL10 Effect on Membrane Integrity.

[0395] Two complementary, dye-based assays will be used to measure
possible CXCL10-mediated increases in membrane permeability as compare to
untreated and CC chemokine controls: propidium iodide (PI) uptake and
diacetyl-fluorescein (DAF) release. PI uptake assays will be performed by
including PI in the treatment sample wells. PI uptake by bacilli, which
correlates to a loss of membrane integrity, will be monitored over a time
course by fluorescence microscopy and/or direct measurement of sample
well fluorescence. For DAF release assays, bacilli will be cultured in
the presence of DAF resulting in uptake and subsequent hydrolysis to
fluorescein, which is stored intracellularly. Supernatants from untreated
and CXCL10-treated samples will be collected, and extracellular
fluorescein released through membrane permeabilization will be measured.
Heat-killed bacilli will be used as positive control for both the PI
uptake and DAF release assays. Untreated bacilli will serve as negative
control.

Site-Directed Mutagenesis of CXCL10 or its Antimicrobial Peptide.

[0396] Working with CXCL10 or a peptide that retains its antimicrobial
activity, we will investigate the effects of generating mutated forms of
the protein/peptide in which one or more lysine residues in the region of
interest (e.g., positively charged C-terminal region) has been
substituted with a neutral amino acid, alanine Initially, substitutions
of 1-3 lysines centrally located in the α-helix will be performed,
assuming that the distribution of these amino acids in the
α-helical turns leave them highly exposed such that they likely
play a key role in charge distribution of the molecules. If the
positively charged C-terminal region of CXCL10 is responsible for its
antimicrobial activity, as predicted by the IL-8 literature, we
anticipate that a C-terminal peptide will retain activity. However, if
the N-terminal region of CXCL10 plays a role in the interaction of CXCL10
with FtsX or other target, then a C-terminal peptide alone may exhibit
reduced or no antimicrobial activity.